Monday, November 21, 2005

A DGGE Guide

DON’T PANIC

A GUIDE TO DENATURING GRADIENT GEL ELECTROPHORESIS

By Stefan J. Green (http://www.stefangreen.com/)
With help from members of the Yahoo DGGE Group
And many other DGGE Users

Version 2 – Updated Nov 28 2005

-- CLICK HERE FOR MOST RECENT UPDATES --

TABLE OF CONTENTS

INTRODUCTION
THEORY
EQUIPMENT REQUIRED
GRADIENT FORMER
PERISTALTIC PUMPS
POWER SUPPLY
PREPARATION OF GELS
Web Protocols (For Bio-Rad)
PCR / NESTED PCR FOR DGGE
DGGE standards
PCR product cleanup prior to DGGE analyses
How much PCR product to load on DGGE?
How much DNA to put in each PCR reaction?
Primer dimerization
Nested PCR
ELECTROPHORESIS
RUN TIME / VOLTAGE
INITIAL VOLTAGE VALUES
AMPERES VS VOLTS
GEL STAINING AND VISUALIZATION
SILVER STAINING
GEL TRANSFER FROM BUFFER TO UV TABLE
ADD GLYCEROL TO INCREASE GEL FLEXIBILITY
GEL ANALYSIS
GEL ANALYSIS PRODUCTS
BAND EXCISION AND ANALYSIS
Avoiding Band Excision by Direct PCR product Sequencing
High-throughput sequencing techniques
HOW TO EXCISE DGGE BANDS
WHAT TO DO WITH YOUR EXCISED BAND
Bead-beating
Gel electrophoresis and cleanup
WHAT TO DO IF YOUR EXCISED BAND ISN’T PURE
A PROBLEMS CHECKLIST
TROUBLESHOOTING AND ADVANCED TOPICS
ACRYLAMIDE PERCENTAGE
BLURRY, FUZZY OR SMEARED BANDS / SUDDEN CHANGE IN QUALITY OF GELS
BUFFER RE-USE
Detection issues / DGGE SENSITIVITY
DGGE ANALYSIS OF CLONES
GC CLAMPS
HIGH DIVERSITY ENVIRONMENTS – MEASURING DIVERSITY
MIGRATION OF BANDS INTO THE GEL IS LIMITED OR ABSENT? (NO VISIBLE BANDS IS A FREQUENT COMPLAINT)
MULTIPLE BANDING FROM SINGLE POPULATIONS AND MULTIPLE SEQUENCES FROM SINGLE POPULATIONS
NON-RIBOSOMAL RNA GENE ANALYSES FOR BACTERIAL COMMUNITY ANALYSIS
PCR ISSUES (There’s no end to these…)
Chimeras / Heteroduplex formation
Degenerate primers
Double bands
PCR Inhibition due to humics and polysaccharides
PCR Contamination
Primer cleanup
Primer Mistmatches / PCR Bias
Primer Design
Quantification
Optimization
General PCR articles
PERPENDICULAR GELS
POLYMERIZATION
SEPARATION OF BANDS
SINGLE STRANDED DNA
SMILING GELS
STORING GELS AFTER ELECTROPHORESIS
WELLS – WAVY, POOR QUALITY, ETC.
DGGE OF HIGH GC PCR FRAGMENTS
FANCY DGGE TECHNIQUES
RECOMMENDED PCR-DGGE PRIMER SETS AND GRADIENT CONDITIONS


INTRODUCTION

Denaturing gradient gel electrophoresis (DGGE) is a commonly used technique in molecular biology and has become a staple of environmental microbiology for characterization of population structure and dynamics. The method is a powerful one, and can rapidly provide a tangible characterization of community diversity and composition, and shifts in population can be readily demonstrated. DGGE analyses are also used in the medical field for detection of mutations, including single nucleotide polymorphisms (SNPs). These advantages are coupled to a number of limitations and these limitations should be well understood before employing the technique. There is often a steep learning curve. The purpose of this page is to provide a source of information for both the beginning and experienced user of DGGE analysis. This site is not meant to be exhaustive, and references to seminal articles will be provided as well as links to other sites with useful information. I would also like to acknowledge the large contribution made by the members of the Yahoo DGGE group (http://groups.yahoo.com/group/dgge/). In large part, this site is a summary of the information provided by members of that community.

While there are a number of trials and tribulations related to the actual operation of the DGGE analysis, it is important to remember that many of the difficulties with DGGE belong to the stages prior to the DGGE. Since DGGE analyses require a significant amount of DNA for detection, a polymerase chain reaction (PCR) must be performed prior to analysis. Thus, all the troublesome features of sampling, DNA (or RNA) extraction, reverse transcription (if employing RNA extraction), PCR primer design, PCR conditions, and PCR cleanup bear some thought when troubleshooting DGGE problems. Every stage of molecular analysis can impact (often negatively) each stage downstream. However, these are considerations that are endemic to molecular biology, and indeed to experimental science as a whole. It has been my experience, however, that DGGE analyses are exquisitely sensitive to PCR problems and thus special attention should be given to this stage. I recommend the following papers for those interested in using DGGE or other any other technique for analysis of microbial diversity, ecology, population structure and population composition:

Morris, C. E., M. Bardin, O. Berge, P. Frey-Klett, N. Fromin, H. Girardin, M.-H. Guinebretière, P. Lebaron, J. M. Thiéry, and M. Troussellier. 2002. Microbial biodiversity: approaches to experimental design and hypothesis testing in primary scientific literature from 1975 to 1999. Microbiol. Mol. Biol. Rev. 66:592-616
von Wintzingerode F, Gobel UB, Stackebrandt E. 1997. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol Rev. 21(3):213-29

The use of DGGE as a tool for analysis of microbial communities has grown in sophistication. It is no longer adequate or appropriate to present DGGE analyses by themselves as an indication of community change. Sequence analyses are the current currency of molecular analyses and must accompany any DGGE analysis. However, DGGE profiles can be analyzed by image analysis software and community profiles as a whole can be taken for cluster analysis. There are a number of features of analysis of complex communities by DGGE which may limit the effectiveness of these cluster analyses; nonetheless, if done properly, such analyses can be powerful. For acquiring sequence information from DGGE, much attention has been focused on the limited sequence information recoverable from the relatively short DNA fragments suitable for DGGE (empirical studies suggest that 500-600 bp fragments are the maximum suitable for DGGE; this has been exceeded occasionally). I will present here various means to recover sequence information from DGGE analyses, and will include a technique for recovering sequence information that is longer than the PCR fragments used for DGGE.

Finally, I would be delighted to receive any comments from the public at large regarding this site and I will be happy to update the site with new information. I can be reached at sjg172@gmail.com or by leaving notes on this site.

THEORY

DGGE analyses are employed for the separation of double-stranded DNA fragments that are identical in length, but differ in sequence. In practice, this refers to the separation of DNA fragments produced via PCR amplification. The technique exploits (among other factors) the difference in stability of G-C pairing (3 hydrogen bonds per pairing) as opposed to A-T pairing (2 hydrogen bonds). A mixture of DNA fragments of different sequence are electrophoresed in an acrylamide gel containing a gradient of increasing DNA denaturants. In general, DNA fragments richer in GC will be more stable and remain double-stranded until reaching higher denaturant concentrations. Double-stranded DNA fragments migrate better in the acrylamide gel, while denatured DNA molecules become effectively larger and slow down or stop in the gel. In this manner, DNA fragments of differing sequence can be separated in an acrylamide gel.

For a full review of theory and of similar methods such as temperature gradient gel electrophoresis (TGGE) or single strand conformational polymorphism (SSCP) the following sites/articles are provided:

· G. Muyzer, E.C. de Waal and A.G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol., 59:695-700. Cited at least 800 times!!!
· Muyzer, G. 1999. DGGE/TGGE a method for identifying genes from natural ecosystems. Current Opinion in Microbiology. 2(3): 317-322.
· Muyzer G and Smalla K. 1998. Application of denaturing gradient gel electrophoresis and temperature gradient gel electrophoresis in microbial ecology. Antonie van Leeuvenhoeck, 73:127-141.
· Liu, W. T., Huang, C. L., Hu, J. Y., Song, L. F., Ong, S. L. and Ng, W. J. 2002. Denaturing gradient gel electrophoresis polymorphism for rapid 16S rDNA clone screening and microbial diversity study. Journal of Bioscience and Bioengineering 93, 101-103.
· Miller, K.M., Ming, T.J., Schulze, A.D. and Withler, R.E. 1999. Denaturing gradient gel electrophoresis (DGGE): a rapid and sensitive technique to screen nucleotide sequence variation in populations. Biotechniques. 27(5):1016-8, 1020-2.
· L.A. Knapp. 2005. Denaturing gradient gel electrophoresis and its use in the detection of major histocompatibility complex polymorphism. Tissue Antigens. 65(3):211.
· D. Ercolini. 2004. PCR-DGGE fingerprinting: novel strategies for detection
· of microbes in food. Journal of Microbiological Methods 56 (2004) 297– 314.
· S. G. Fischer and L. S. Lerman. 1983. DNA Fragments Differing by Single Base-Pair Substitutions are Separated in Denaturing Gradient Gels: Correspondence with Melting Theory. PNAS 80:1579-1583.
· Glavac D, Dean M. 1993. Optimization of the single-strand conformation polymorphism (SSCP) technique for detection of point mutations. Hum Mutat. 1993;2(5):404-14.
· Sheffield VC, Beck JS, Kwitek AE, Sandstrom DW, Stone EM. 1993. The sensitivity of single-strand conformation polymorphism analysis for the detection of single base substitutions. Genomics.16(2):325-32.
· Zhongtang Yu and Mark Morrison. 2004. Comparisons of Different Hypervariable Regions of rrs Genes for Use in Fingerprinting of Microbial Communities by PCR-Denaturing Gradient Gel Electrophoresis. Appl. Environ. Microbiol. 70:4800-4806.
· Melt-Madge (Thermal Time Ramp Gel Electrophoresis http://www.ngrl.org.uk/Wessex/meltmadge.htm

Some users may also want to consider terminal restriction fragment length polymorphism (T-RFLP) analyses for community structure analyses. The advantage of such analyses is that they can provide some phylogenetic information based on restriction fragment analysis, and such analyses can also generate some quantitative data that is less readily available with respect to DGGE analyses. In addition, T-RFLP may be suitable for some functional genes and gene fragments sizers for which DGGE analyses are not suitable. T-RFLP may also avoid some problems with degenerate primers that can plague some DGGE analyses. Some manuscripts and websites to consider below:

· Beatriz Díez, Carlos Pedrós-Alió, Terence L. Marsh, and Ramon Massana. 2001. Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques. Applied and Environmental Microbiology, 67(7): 2942-2951.
· Osborn, A. Mark, Moore, Edward R. B. & Timmis, Kenneth N. 2000. An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environmental Microbiology 2 (1), 39-50.
· S.L. Dollhopf, S.A. Hashsham, J.M. Tiedje. 2001. Interpreting 16S rDNA T-RFLP Data: Application of Self-Organizing Maps and Principal Component Analysis to Describe Community Dynamics and Convergence. Microbial Ecology 42(4): 495 – 505.
· Frag Sorter by Fred Michel: http://www.oardc.ohio-state.edu/trflpfragsort/contact.php

EQUIPMENT REQUIRED

I do not explicitly consider the upstream equipment requirements for DGGE (i.e. PCR machines), though I can highly recommend a temperature gradient PCR machine. When applying new primers to an environmental system, having a temperature gradient PCR can be extremely useful, and coupled with DGGE analyses, one can rapidly assess a variety of annealing temperatures.

Before diving into various links to the dominant DGGE manufacturers, I would like to comment that there is a new technology which works in a similar manner, but employs an HPLC column rather than an acrylamide gel for separation. I have not tested this out myself, but the instrument has several theoretical advantages over DGGE analyses, namely that no DGGE gels have to be poured, and that a fraction collector can be employed to recover various “bands”. For those interested, see the following website: www.transgenomic.com/ (denaturing HPLC).

Essentially a DGGE system is a heated fish tank that you can run voltage through. The critical issues for DGGE systems are temperature control and stability of current. There are several major DGGE manufacturers in the market. These include, but are not limited to, the following:

Bio-Rad
http://www.blogger.com/Local%20Settings/Local%20Settings/Temporary%20Internet%20Files/Local%20Settings/Temporary%20Internet%20Files/OLK1B7/www.bio-rad.com
Ingeny
http://www.ingeny.com/
http://www.ingeny.com/ingeny_brochure_eng.pdf
CBS Scientific
http://www.cbssci.com/

I have personal experience with both the Bio-Rad and the Ingeny. I believe that the CBS Scientific is probably the least expensive of the three and the Ingeny the most expensive. The Bio-Rad is probably the most commonly used, and while you can get very nice gels from this machine, there are a number of recurring problems, including:
o poor quality back gels (due to proximity to heating element, lack of adequate stirring)
o easily broken glass plates/spacers/clamps/heating element (all which can be replaced at an expensive cost)
o need to remove the active, heavy part of the DGGE system from the buffer to put gel in, remove gel, add samples, etc (this increases the likelihood of breaking the heating element, and cooling of the buffer during loading and cleaning of gels)
o limited number of lanes and edge smiling (all DGGE systems get this)
o Corrosion of electrodes
o Awful gradient-forming device (I recommend purchasing a different gradient-former).

The Ingeny system, in my experience, has many advantages over the Bio-Rad system, but it also has a few annoying quirks. These advantages and disadvantages:
o No stirring (instead, a re-circulating pump – this also helps with cleaning lanes)
o No need for removal of heating element from buffer tank (this allows heating during cleaning and loading of gel)
o U-shaped spacer eliminates the need for pouring a seal at the bottom of the gel. However, when greasing the spacers (to reduce smiling) the spacers can become warped and are not easy to push down. I have also found that getting rid of bubbles trapped between the bottom of the gel and the “pushed down” U-shaped spacer is the most annoying feature.
o A very large buffer volume (17 L) which probably helps with temperature stability, but is somewhat annoying in terms of buffer preparation.
o Large gels allow 32 lanes (routine) and 48 lanes (very narrow)
o Comes with a reasonable gradient former

I cannot currently comment on the CBS system, but if someone would write in, I would be delighted to add some comments. I have heard that it also has a large buffer volume (18 L) and can hold 4 gels.

In addition to the DGGE machine itself, you will need a:

GRADIENT FORMER

The simplest and most effective design for a gradient former can be purchased from a number of different manufacturers, but I have managed to track down the website location for those from CBS Scientific. The largest gels, as far as I know, are for the Ingeny and have a total volume of about 50-60 ml. These therefore require the CBS Scientific Cat. # GM-100 – which has a total volume of 50 ml on each side.

You will also require a small stir bar (Teflon coated) to stir one chamber of the gradient former, a stir plate, a peristaltic pump (see below), and teflon or similar tubing.

PERISTALTIC PUMPS

http://www.rainin-global.com/rp1.htmlhttp://www.lplc.com/instruments/pump.htmhttp://www.millipore.com/catalogue.nsf/docs/C419

POWER SUPPLY

Any reasonable power supply should serve. It should be able to run 200 V, and up to 300 V may be useful for some users.


PREPARATION OF GELS

Each system will have slightly different preparation methods and you should follow the recommended protocols as best you can. I have attached links to a few web-published protocols. In addition to the equipment listed above, you will need the following items. I highly recommend that you purchase the products already made (e.g. 40% acrylamide solution rather than making your own from powder; buying deionized formamide rather than using a resin , etc):

· A front and back glass plate
· 1 mm spacers (U-shaped spacers for Ingeny system)
· Spacer Grease [I use Dow Corning High Vacuum Grease, but others should be suitable] HIGHLY RECOMMENDED to reduce gel smiling at perimeter. Coat the spacer with a thin layer of grease and wipe off excess grease with a kimwipe.
· Appropriate comb
· Gel casting station
· 40% Acrylamide (37.5: 1, acrylamide:bis-acrylamide)
· Formamide (deionized)
· Urea
· 50X TAE (50X is 242g Tris base; 57.1 ml glacial acetic acid; 100 ml 0.5M EDTA (pH 8) per liter)
· Ammonium Persulfate [Is highly hygroscopic – remember to buy fresh routinely]
· TEMED (N,N,N',N' -tetramethylenediamine)
· Nucleic acid stain
o GelStar
o EvaGreen
o Ethidium Bromide [Not recommended due to low sensitivity, toxicity]

o Prior to casting of the gels, you will need to have made stock solutions of a zero denaturant acrylamide solution and a high denaturant acrylamide solution (I generally make 80% solutions, defined below).

The zero denaturant acrylamide solution will vary slightly according to final acrylamide solution, but a 100 ml stock solution of 6% acrylamide, zero denaturant solution will contain 15 ml of 40% acrylamide (37.5: 1, acrylamide:bis-acrylamide), 2 ml of 50X TAE, and water to 100 ml. A 100 ml stock solution of 6% acrylamide, 80% denaturant will contain 15 ml of 40% acrylamide, 33.6g of Urea, 32 ml of formamide, 2 ml of 50X TAE and water to 100 ml. This can be a bit tricky to make. I usually add all the liquids together first (acrylamide, formamide, TAE and a bit of water) and then add the Urea. This can take a while to get into solution and mild heating can help, though it shouldn’t be necessary. Not to be repetitive, but many of these compounds are highly toxic. Higher acrylamide concentrations can be even trickier when making the high denaturant solution – be careful not to add too much water.

Prior to casting of the gel, specific concentrations of denaturing solutions can be made by mixing the zero and high denaturant solutions in appropriate ratios. I prefer to make the solutions by mixing integer values of each solution (therefore, no 6.35 ml etc). This will get you reasonably close to the value you want (for example, for the Ingeny system I used 16 ml of zero denaturant and 6 ml of 80% denaturant solutions to get a 22% solution). Since you should be pouring a gradient that is wider than the region you want, a little variation at either extreme is not particularly significant.

I make fresh APS (10% solution, in water) fresh before each gel. However, aliquots can be frozen and used for some time (ca. 1-2 wks). In general, fresher reagents are better and a number of problems with DGGE gels can be traced to old acrylamide, formamide and TEMED solutions. These solutions are certainly a place to start when trying to trouble shoot. Nonetheless, I have found that acrylamide solutions, kept at 4C, can keep for months without any negative effect. Well cleaned glass plates also help improve gel quality. Some DGGE operators clean the glass plates with ethanol as a last stage; I find that this often diminishes the quality of the gel pouring.

Web Protocols (For Bio-Rad)

Protocol from the Laboratory for Microbial Ecology Department of Earth, Ecological and Environmental Sciences University of Toledo
http://www.eeescience.utoledo.edu/Faculty/Sigler/RESEARCH/Protocols/DGGE/DGGE.pdf

G. Zwart and J. Bok, dept. of Microbial Ecology, Center for Limnology, Netherlands Institute of Ecology (NIOO-KNAW). E-mail: http://www.blogger.com/Local%20Settings/Local%20Settings/Temporary%20Internet%20Files/Local%20Settings/Temporary%20Internet%20Files/OLK1B7/Zwart@cl.nioo.knaw.nl
http://www.kuleuven.ac.be/bio/eco/bioman/publications/Protocol-DGGE_bacteria&protists.pdf

Well, I haven’t found any online protocols for Ingeny or CBS (I haven’t looked very hard), but pouring these gels is essentially the same as any other gel.

PCR / NESTED PCR FOR DGGE

DGGE standards
There are no established standards for DGGE. However, applying homemade standards to DGGE gels can be very useful. For some gel analyses programs, it is useful, if not essential, to have standards every few lanes or so due to the heterogeneity of the DGGE gels. I have seen some users load size standards onto DGGE gels, though these don’t have much meaning. The best approach is to create your own set of standards by mixing PCR product of a number of differently migrating clones. You should run the PCR of each clone independently and then mix the PCR yield. Make a large stock.

In addition, you may want to check out a new technique of adding standards to each lane. This can be found in Section 8.0 Gel Analysis. See the article by Neufeld and Mohn, 2005.

PCR product cleanup prior to DGGE analyses
There has been some discussion in the Yahoo DGGE group as to whether or not it is useful to cleanup PCR products prior to DGGE analysis. I have never routinely performed PCR cleanups and I think it would be a major added expense and effort. Luckily, there doesn’t seem to be any major reason to cleanup the PCR product. The primers and primer dimers migrate out of the gel and do not interfere with final staining analyses. However, you may need to cleanup the PCR product if you want to do cloning or quantification.

How much PCR product to load on DGGE?
There is no established amount of PCR product to load on DGGE. However, some things to consider: the lower the diversity your system, the less DNA you need to load. For example, if you load 50 uL of DNA from a PCR of a single clone, you will definitely load too much DNA. However, in a highly diverse microbial community, that PCR product will be split among a large number of different sequences, making detection of some problematic. Another rule of thumb suggests that if (ignoring for the moment any issues with PCR bias) a sequence is not at least 1% of the total population targeted by the primer set, that sequence will be difficult to detect by DGGE. Thus, specific primers can aid in detecting less abundant populations by narrowing the range of target population.

From the DGGE group I have seen suggestions that 300-500 ng of DNA is an appropriate amount to load per lane – for an environmental sample, or more specifically, 2-20 ng per band, when using non-EtBr stains. Obviously, for PCR product of a pure culture or clone, you’ll need significantly less. Although not widely done, it probably would be a good idea to quantify PCR products prior to loading on DGGE gels, so as to be able to compare intensities between lanes. If you have weak PCR, you can load large volume into each lane by loading twice. That is, fill the well, electrophorese for a while and the load the rest of the PCR product. Since DNA fragments often stop when they reach their specific denaturant concentration, and run times are probably longer than necessary, there does not appear to be any negative effect resulting from sequential DNA addition.

One thought – the size of the PCR fragment may also be a consideration. Given the same copy number, a longer DNA fragment will yield a stronger fluorescent signal.

How much DNA to put in each PCR reaction?
This topic has come up recently in the Yahoo group. Ideally, a similar amount of DNA should be added to each PCR reaction, but in reality, this is often time consuming to do. This requires the measurement of DNA concentration from each DNA extract, and then dilution of each DNA to the same final concentration. This may be particularly important for quantitative PCR. For non-quantitative PCR of environmental samples ultimately used for DGGE, there seems to be a range of 10 to 100 ng (per 50 microliter reaction) in use. I have seen on the DGGE Yahoo group that some users have been diluting genomic DNA prior to adding it to PCR. Diluting DNA to avoid contaminants such as humic acids/polysaccharides may work sometimes, but in general, it is probably better to clean up the DNA, even if you loose a proportion of your DNA. The article below suggests that diluting DNA makes reproducibility worse.
· Chandler DP, Fredrickson JK, and Brockman FJ. 1997. Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries. Molecular Ecology 6: 475-482

Some situations can be far more complex. As Kim discusses in post #1267, one can have a situation where there is a variable amount of non-target genomic DNA. Thus, it may not be entirely fair to add the same total amount (ng) of genomic DNA to each PCR reaction, if the amount of target DNA varies from sample to sample. The example given is that of DNA extracts from plant samples. If one is looking at microbial communities associated with plant roots, one might recover variable amounts of root DNA depending on growth stage, etc. I don’t have a specific answer to the question of whether or not one should adjust the amount of genomic DNA added to PCR reactions based on the knowledge that variable amounts of non-target DNA are present. It seems likely that in many environments there will be variable amounts of non-target DNA (e.g. fungal DNA in bacterial population analyses, etc.) and that these cannot be readily quantified. Since standard PCR is not overly sensitive to starting concentration (thus the requirement for real-time PCR) this may not be something to worry about too much.

Primer dimerization
Some PCR primers form strong primer dimers than can be visualized by agarose electrophoresis [See figure below]. I have not found that these interfere with DGGE analyses as they appear to migrate out of the gel. However, these primer dimmers can be a problem if you run ligation/transformation/sequencing reactions directly from the PCR product. The high quantity of the primer dimer can decrease the efficiency of the cloning reaction because the dimer will compete for plasmid. This is because the primer dimers are double-stranded, and are A-tailed (if you are using Taq polymerase) just as the PCR product of the correct size. Since these primer dimers can be extremely abundant, and are double-stranded and a-tailed, they can compete with the primary PCR product in the ligation reaction. Thus, many of the clones will contain only the primer dimers, decreasing the efficiency of the ligation/transformation reaction. Thus, you may want to clean up your PCR product prior to cloning. Remember, if a band of smaller size (bp) is of the same intensity as a band of larger size, there are actually a greater number of copies of the smaller fragment.

Nested PCR
Nested PCR is a technique by which genomic DNA is subject to PCR amplification and then the product resulting from the amplification is subject to a second, nested PCR with primers that target a region within the region targeted by the first PCR primers. There are a number of reasons to employ nested PCR:
· Increase PCR yield from weak reactions
· Avoid detrimental effects of PCR amplification with primers that have a GC-clamp
· Generate specific DNA fragments suitable for DGGE analysis from DNA fragments that are not suitable for DGGE analysis
· Allow direct DGGE comparisons of general and specific population analyses
· Recover additional sequence information than that provided in DGGE-appropriate fragments.
· Avoid having to develop and optimize new DGGE conditions for a new primer set.
· Compare the community recovered by different primer sets (for example - PCR large fragments with different general bacterial primer sets and then nest with the same general bacterial primer sets and compare by DGGE. This is limited by the internal primer set, but it can still reveal important differences in primer sets.)

I have added a small flow chart below to show some of the potential. I think that in particular, the capacity to generate sequence information that is larger than the DGGE fragment is underutilized.

There are some caveats related to nested PCR. Namely, since there are multiple PCR reactions and high numbers of PCR cycles, the potential for chimera formation and sequence errors increase. Also, you will find that nested PCR can often generate secondary, non-specific bands. You should optimize PCR conditions (temp, cycle #, magnesium concentration) to reduce the intensity of these bands, if possible. All things being equal, if you can achieve your aims without nested PCR, you probably should avoid it. However, for some things, the nested PCR approach is so useful, it’s worth it. If you are worried about the quality of sequences recovered from nested PCR, you can apply specific primers (developed based on the sequence information recovered from the nested PCR approach) to the same samples to demonstrate that the sequences are actually present.

When I conduct nested PCR, I usually dilute the original PCR product 1:5 or 1:10. I do not perform a cleanup prior to the second stage of PCR. However, I generally raise the annealing temperature of the second stage of PCR by 4C relative to the standard temperature for the given primer set (this will also help avoid contamination of the blank, by the way…), and I perform fewer cycles and often I add less primers. These are all attempts to limit the efficiency of the PCR reaction so as to not get too many secondary, non-specific bands.

· Shabir A. Dar, J. Gijs Kuenen, and Gerard Muyzer. 2005. Nested PCR-Denaturing Gradient Gel Electrophoresis Approach To Determine the Diversity of Sulfate-Reducing Bacteria in Complex Microbial Communities. Appl. Environ. Microbiol. 71:2325-2330.
· Wood GS, Uluer AZ. 1999. Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE): sensitivity, band pattern analysis, and methodologic optimization. Am J Dermatopathol. 1999 Dec;21(6):547-51.
· Green, S.J., Freeman, S., Hadar, Y. and Minz, D. 2004. Molecular tools for isolate and community studies of Pyrenomycete fungi. Mycologia, 96(3), 2004, pp. 439-451.


ELECTROPHORESIS

· Vanessa M. Hayes, Ying Wu, Jan Osinga, Inge M. Mulder, Pieter van der Vlies, Peter Elfferich, Charles H. C. M. Buys, Robert M. W. Hofstra. 1999. Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis. Nucleic Acids Research. 27(20): 29.

RUN TIME / VOLTAGE
There has been considerable discussion on the yahoo group about the correct run times and voltages. In general, longer run times with lower voltages tend to produce better quality gels. Voltages are in the range of 50-250V, and run times are generally from 3 hr to 17 hr. A standard run time of 17 hr (essentially overnight) at 100 V produces high quality gels for many PCR fragments. The exact voltage may differ between PCR product size, buffer concentration, buffer temperature, run time, and gel acrylamide percentage. For very short fragments, it may be important to use relatively low voltages for a long run to avoid having the fragments migrate out of the bottom of the gel. For gels in which the fragments completely stop within the gradient, extra run time will probably not effect the final gel. It seems that Volt-Hours are probably the best thing to consider. We used to run some gels at 250 V for 3 hr and found 17 hrs at 100 V yielded better gels; the 100V run had 1700 volt-hrs while the 250 V run had only 750. It should be noted that if you are particularly worried about running your fragments out of the gel, there may be a problem with your gradient (i.e. not wide enough) or your acrylamide concentration (not high enough). Every user will have a preferred voltage and run time, so I am not going to make any absolute recommendations here – there appear to be many possible conditions which yield good results. Many times the conditions will have to be empirically determined.

INITIAL VOLTAGE VALUES
Some users will run a high voltage initially to bring the DNA into the gel and then use a lower voltage for the rest of the run. In the Ingeny system, one is supposed to run the gel for a little while to draw the DNA into the gel before turning on the recirculating pump – otherwise the pump may stir up the PCR product in each well. I do not think that running an initial high voltage is important; the critical factor is to have the temperature stabilized. With the Bio-Rad, you have to keep removing the heating element from the tank and during the cleaning of the wells and loading of samples, the buffer cools down. Before starting the electrophoresis, you should make sure that the temperature of the tank is at 60C (or whatever temperature you are using). You can run a low voltage while the tank is heating to its final temperature just so you limit diffusion of the DNA into adjacent wells. The important thing is to not let the DNA migrate into the gradient until the buffer is at the correct temperature.

AMPERES VS VOLTS
I have seen a lot of discussion about voltage vs amperage when dealing with electrophoresis. You could run your electrophoresis with either fixed voltage or amperage, but the standard appears to be constant voltage. I have found that when running gels with a fixed voltage, the resulting amperage is an excellent indicator for problems with the system. Old buffer, leaks in the system, air bubble blocking the bottom of the gel, poor circulation, temperature – all these factors can affect the operation of the system and these will show up in the amperage – usually by low amperage relative to a well operating system. For example, on my Ingeny DGGE system, I can expect approximately 40 mA when running a single gel at 100V, 60C. To make sure my system is properly set up prior to loading samples, I run a few minutes of electricity through the system at 100V to make sure I get 40 mA (obviously each system will have a different “standard” mA value at a fixed voltage). If the values are way off I know there is some sort of problem. I never load my samples until the mA values are within proper ranges. You should also remember that at the same voltage, running 2 gels will yield a higher mA than running 1 gel. Regardless of how many gels you are running, you should maintain the same voltage. If your gel box cannot handle the amperage from multiple gels, you may have to run at a lower voltage for a longer time.

GEL STAINING AND VISUALIZATION
Do NOT add stain to the gel prior to electrophoresis. Staining of DGGE gels MUST be done after electrophoresis. After stopping the electrophoresis and having separated the gel plates, leaving the gel attached to one plate, you are ready to stain the gel. There are a number of nucleic acid dyes which are adequate for visualization of DGGE gels. I recommend not using Ethidium Bromide (EtBr). EtBr is a strong mutagen and has a much lower sensitivity that some of newer nucleic acid dyes available (listed above). EtBr also cannot be excited well by wavelengths above 400 nm. This is a major downside if you want to use non-UV illumination tables (see below).

I am currently using GelStar as a nucleic acid stain. It is somewhat expensive, but not prohibitively so, is less toxic and more sensitive that EtBr. I recommend staining while shaking for 30 minutes and then transferring to a de-staining tank (water is fine) for another 15-30 minutes. GelStar and similar stains will be useful if you want to use a non-UV illumination table. I can recommend the Dark Reader illumination table : http://www.clarechemical.com/. This product excites the nucleic acid dyes at wavelengths from roughly 400 – 500 nm. Then a second filter is placed over the gel which blocks out light below 500 nm. Since the dyes emit at wavelengths above 500 nm, the gels can be visualized. The advantages of this table are that there is no UV light, and no damage to the DNA in the gels. If you are intending to excise bands from DGGE gels, I can recommend this table. There is no hurry to excise bands without doing damage to the DNA in your gel or in your body, and you can place the gel, with the glass plate, onto the table. The quality of the pictures from this table is not as good as a UV table. Thus, for publication quality photos, you’d better have a UV transilluminator. The Dark Reader also comes with goggles that contain the second filter. Thus you can easily excise bands without any filter in the way.

SILVER STAINING
I’m not sure anybody still silver stains, but here’s a reference. I remember some users had problems extracting bands and re-PCRing after silver staining. One advantage is that the signal does not degrade rapidly as with some of the newer stains.
· D. Radojkovica and J. Kusic. 2000. Silver Staining of Denaturing Gradient Gel Electrophoresis Gels. Clinical Chemistry 46: 883-884.

GEL TRANSFER FROM BUFFER TO UV TABLE
Transferring the gels from the staining bath to the UV table can be a tricky and annoying procedure. I have seen published papers with 5 or 6 DGGE gels and every single one of the gels is ripped somewhere. Handling the thin gels, particularly the 6% acrylamide gels, takes experience. I previously posted some tips (see below) on the Yahoo DGGE group, and the best tip is to keep everything lubricated with buffer or water – this reduces friction and tearing.

The best word is patience. Do everything slowly and make sure there is plenty of liquid around to lubricate the transfer of the gel. You can reduce your anxiety by removing one step - when you transfer the gel to staining, keep the gel on the bottom plate and simply put the whole plate in the staining bath. I shake the gel off the bottom plate while it is in the staining bath to make sure that the dye can diffuse in from all directions. When lifting the gel out of the staining bath (and then into a de-staining bath and/or directly onto the UV table) lift the plate out of the staining buffer partially - this allows you to see where the gel is. If the gel is centered on the plate, place one palm on top of the gel to keep it in place while you lift the whole plate out (with the gel) using the other hand. Let some of the liquid drain off the plate (keep your palm on the gel). Now place the plate on your UV table at an angle and let the gel slide onto the table (gently, slowly, patience....); as the gel comes off slide the plate further away. Have a water bottle available and add water when needed. Also, you may have to nudge the gel if it gets stuck - particularly at the edges where there is grease (from the spacers). Don't worry about having the gel be perfect as it slides off the glass plate. It can be adjusted easily on the UV table with gentle nudging and more water.

ADD GLYCEROL TO INCREASE GEL FLEXIBILITY
Dr. Von Sigler of the Laboratory for Microbial Ecology (University of Toledo) has this to say about glycerol: “ I add glycerol to a final concentration of 2% (v/v) because it adds a great amount of flexibility to the gels. This added flexibility comes in handy when manipulating the gels (removal from the plates, staining, placement and positioning on transilluminator) and decreases the risk of tearing the gel. It does not impact DNA migration or the banding qualities.”

GEL ANALYSIS
I cannot claim to be a great expert on dendogram-style analyses of DGGE gels. I am going to list some known programs, some articles and hope for some contributions from more knowledgeable users. Some pitfalls to be considered, however, are the problem of multiple populations co-migrating to a single band position, multiple bands from a single organism, and PCR artifacts resulting from degenerate PCR primers. In addition, if the dendogram analyses also consider the intensity of each band, then it should be important to either load the same amount of DNA in each lane, or to make intensity values relative to total intensity. Some of the commercially available programs can be extremely expensive.

I highly recommend a new manuscript in AEM by Josh Neufeld and William W. Mohn (“Fluorophore-labeled primers improve the sensitivity, versatility, and normalization of denaturing gradient gel electrophoresis”). This manuscript develops a new technique to increase our capacity to use DGGE gels for digital analysis by including standards within each lane. These standards have fluorescent tags that fluoresce at different wavelengths than the fluorescent molecules attached to the unknown PCR product. Brilliant! The caveat is as the authors note, besides expense, “that access to an expensive laser-scanning instrument is required, which may limit widespread use of this application at this time.” One other thing: since the sensitivity is increased by this method, the authors could reduce the number of PCR cycles required to generate enough DNA for DGGE. This is another advantage.

· Neufeld, JD and WW Mohn. 2005. Fluorophore-labeled primers improve the sensitivity, versatility, and normalization of denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 71:4893-4896.

Jaak Truu of the Yahoo DGGE group has this to say: “Standard ordination methods (PCA; CA) do not take into account the situation that one band may represent several species. There are some similarities between data obtained with DGGE and microarrays (noisy data). In case of microarray data new methods and software applications are developing very fast. For DGGE there are some papers considering in depth statistical analysis of microbial community fingerprints (Wilbur et al., 2002 [See Below]). The simplest way (but not only one) to confirm the grouping or clustering of your DGGE data obtained with cluster analysis or ordination is use methods that implement bootstrapping, randomization or MonteCarlo methods. Unfortunately these methods are not included in generally used statistical analysis software.”

· Tong Zhang and Herbert H.P. Fang. 2000. Digitization of DGGE (denaturing gradient gel electrophoresis) profile and cluster analysis of microbial communities. Biotechnology Letters. 22: 399 – 405.
· Fromin, N., Hamelin, J., Tarnawski, S., Roesti, D., Jourdain-Miserez, K., Forestier, N., Teyssier-Cuvelle, S., Gillet, F., Aragno, M. & Rossi, P. 2002. Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns. Environmental Microbiology 4 (11), 634-643.
· J. Wilbur, J.K. Ghosh, C.H. Nakatsu, S.M. Brouder, and R.W. Doerge. 2002. Variable selection in high-dimensional multivariate binary data with application to the analysis of microbial community DNA fingerprints. Biometrics 58:378-386.

GEL ANALYSIS PRODUCTS
· BioNumerics (Applied Maths, Kortrijk, Belgium)
· Quantity One (Bio-Rad)
· DGGESTAT
· Gelcompare II
· Bioprofil (Vilbert-Lourmat)
· Jayson D. Wilbur from Purdue University has written “There is a free statistical software package called "R" that can be used to construct dendograms based on the Ward method. It is available at: http://www.r-project.org/ You will also need to download the "cluster" package from the website and use the commands "agnes" and "plot.agnes" (specifying the option method="ward").”

BAND EXCISION AND ANALYSIS
This subject is probably one of the most frequently discussed on the Yahoo DGGE group. Having done many, many band excisions, I cannot say that I find it particularly enjoyable and I believe, as I mentioned on the Yahoo Group (see below), that it is not the best way to go about acquiring sequences. However, for bands of particular interest that cannot be acquired in any other manner, it should be used.

Avoiding Band Excision by Direct PCR product Sequencing
The choice of bands to excise may also be highly subjective, and, as many of us have experienced, there can be multiple sequences hiding in a single band. Thus, excising, cloning, and sequencing of a single clone from each band can miss hidden diversity. Furthermore, excision of bands is time consuming, and if done on a UV table, unpleasant and can be damaging to DNA (yours and your sample's). If you want to be sure that the excised band is the correct thing, you should re-PCR, run another DGGE, clone the PCR product, and then screen the clones with another DGGE analysis. All of this work makes me wonder - what is the point? I would like to suggest that a better way to approach this is to clone directly from the original PCR product (using GC-clamped PCR primers) and then screen the clones against the environmental sample. Thus, from a single cloning reaction you will be able to pick off many of the dominant bands and you will only have to run a single DGGE instead of 3. I would suggest that excision of bands should only be done in those cases where important bands simply cannot be recovered by an initial cloning reaction. In addition, at least 2 clones for each band position should be sequenced - this can help verify if there is hidden diversity at each band position.All of this cloning would have been an onerous burden. However, there are now sequencing facilities that will take clones in 96-well plates, extract plasmid and run sequencing reactions for approximately $3/rxn [NOTE: this is in the year 2005]. This means that you can avoid having to do plasmid extractions yourself and that the cost will still be less than you used to pay. So, perhaps we should pick more colonies, clone more and excise bands less.

High-throughput sequencing techniques

· Neufeld, JD, Yu, Z, Lam, W, and WW Mohn. 2004. Serial Analysis of Ribosomal Sequence Tags (SARST): a high-throughput method for profiling complex microbial communities. Environ. Microbiol. 6:131-144.
· Kysela, David T., Palacios, Carmen & Sogin, Mitchell L. 2005. Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition. Environmental Microbiology 7 (3), 356-364.
· Yu, Zhongtang, Yu, Marie & Morrison, Mark. 2005. Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition. Environmental Microbiology

HOW TO EXCISE DGGE BANDS
You can excise DGGE bands with two basic approaches. The first is to take a sterile pipette tip by hand and stab it into the gel at the position of the band that you are interested in. Upon removing the tip, you then inoculate a fresh PCR reaction with the DNA adhering to the tip. I have had very mixed results with this method and do not recommend it. While it is certainly time saving, in a sense, you often do not get the band that you are interested in.

The second technique is to physically excise the band with a sterile razor blade. You should be careful to limit your exposure to UV (if you are using a UV transilluminator), and there is a limit to the amount of exposure your DGGE gel can get before the DNA is significantly damaged and not particularly usable for downstream analyses. Both these techniques can be difficult when the bands are very close together. In all cases, it is critical that the PCR product from this re-amplification be screened against the environmental sample on a second DGGE analyses to ensure that the PCR product reflects the excised band.

WHAT TO DO WITH YOUR EXCISED BAND
Bead-beating
Place the acrylamide fragment in a tube with some glass beads and water (or TE) and bead-beat or vortex briefly. Then incubate the tube either at 37C for 30 min, or 4C for several hours/overnight. A microliter or two of the water can then be used as a template for a subsequent PCR reaction. I would centrifuge the tube prior to taking liquid for the PCR reaction to remove any possible acrylamide pieces. Remember not to overload the PCR reaction with DNA – this can cause subsequent problems with smearing on DGGE gels.

Gel electrophoresis and cleanup
This is a clever technique to transfer the DNA in the acrylamide to agarose. The agarose can then be melted and the DNA recovered. Place the acrylamide fragment into a well in an agarose gel and fill the well with fresh liquid agarose. Allow the agarose to cool and gel, and then electrophorese the gel. The DNA will migrate out of the acrylamide and into the agarose gel. The fragment can then be excised from agarose and cleaned up either with a DNA gel cleanup kit or a sodium iodide/silica cleanup (see below):
· Boyle JS, Lew AM. 1995. An inexpensive alternative to glassmilk for DNA purification. Trends in Genetics. 11(1):8.

If you re-PCR the excised DNA, you will definitely need to screen the PCR product against the original environmental sample with a second DGGE. Alternatively, you can clone directly, and then screen the clones against the environmental sample. Under no conditions is it a good idea to clone directly and send for sequencing without verifying that the clone band is reflective of the environmental band that you were interested in.

You may want to use one of your PCR primers for sequencing analyses, even if you have cloned your PCR product. I recommend using the primer without the GC-clamp. In that manner, the sequencing reaction begins at the end of the fragment without the GC clamp, and you don’t waste the best stage of the sequencing reaction on the GC clamp region. If your fragment is short enough, you probably don’t have to worry.

WHAT TO DO IF YOUR EXCISED BAND ISN’T PURE
This is a common enough problem that has no particularly easy solution. When excising bands, or stabbing bands into PCR, it is easy enough to pick up unwanted DNA. Thus, screening of excised bands, cloning, and screening of cloned bands are all part of the work required to ensure that you have isolated the correct band. Even if an excised band isn’t pure –that it, a mixture of several bands, it can often be much less diverse than the original sample and can help you isolate the band of interest. Remember, if you are going to clone the excised band (highly recommended), you simply need to get the ratio of target:non-target high enough to make it likely that you will recover the band of interest by cloning.

A PROBLEMS CHECKLIST

Ø Back gel on Bio-Rad DGGE is often of poor quality perhaps due to inadequate mixing and proximity to the heating element.
Ø Are you using the Bio-Rad wheel to cast gels? Get yourself a gradient former (see above).
Ø Did you clean the wells properly? Unpolymerized acrylamide and urea and formamide diffusing upwards can cause problems. Clean wells thoroughly before loading.
Ø Did you grease the spacers? This can help reduce smiling on the edges.
Ø Are there air bubbles in your gel? Try to avoid air bubbles getting trapped in your gel during pouring. Tap on the glass to cause the bubbles to rise to the surface. Bubbles in gels can cause streaking in the lanes. Avoid air bubbles getting trapped under the comb by inserting the comb at an angle and slowly.
Ø Are your reagents fresh? Many problems have been traced to old or poor quality reagents both for DGGE and for PCR. dNTPs, acrylamide, and formamide have repeatedly come up as the culprit in poor quality DGGEs on the Yahoo DGGE Group.
Ø Are your reagents high quality? Molecular Biology grade chemicals should be used.

TROUBLESHOOTING AND ADVANCED TOPICS

ACRYLAMIDE PERCENTAGE
You will have to determine what acrylamide solution is correct for your system and it may vary according to primer set. For most environmental DGGE analyses 6% acrylamide is used. Shorter fragments may use an 8% acrylamide solution and in some cases an acrylamide gradient can also be included. I have used acrylamide solutions of up to 12% (rare) [See below].
· Green, S.J., Freeman, S., Hadar, Y. and Minz, D. 2004. Molecular tools for isolate and community studies of Pyrenomycete fungi. Mycologia, 96(3), 2004, pp. 439-451.

I have found with one primer set generating a PCR product of about 400 bp, that an 8% gel gave much worse results that a 6% gel. I was really surprised – I had been running the primer set at 8% acrylamide for a long time and had reasonable results, but limited visible diversity. By chance I ran the sample PCR product on a 6% gel and got amazingly higher diversity and better separation. I think ultimately empirical tests are required when optimizing any new primer set for DGGE analysis. However, it seems reasonable to think that small fragments would be better off with higher acrylamide percentages.

BLURRY, FUZZY OR SMEARED BANDS / SUDDEN CHANGE IN QUALITY OF GELS
These types of problems are often a sign of PCR problems. However, they can also indicate a poorly made gel, poor quality or old reagents, an improper gradient, irregular current, old buffer, temperature control issues (e.g. back-gel issues on the Bio-Rad), etc. Also, you may want to consider if your reagents, even if fresh, are really good. I have been satisfied with Bio-Rad reagents and Sigma reagents as well. It is best to be consistent once you have found good reagents.

If your problem can be traced to using the back gel on the Bio-Rad machine, you have a couple possible solutions:
>> Don’t use the back gel
>> Place the entire apparatus in a water bath heated to the same operating temperature. This will help the system maintain temperature control better.
>> Place a stir bar in the bottom of the tank and put the entire apparatus on a stir plate to increase mixing of the system. Be careful – some stir plates cannot handle the weight from the full Bio-Rad system (This is from personal experience….).

Bands in the top of the gel are often fuzzy and indistinct, and this may be an indication that these are some sort of artifact of the PCR/DGGE analysis. In particular, if I notice a band that is present in all samples, very high in the gel, and fuzzy, I am highly suspicious of it. These can be heteroduplex bands which denature rapidly, or perhaps single stranded DNA.

For sudden change in gel quality:
>> Check primer quality; re-order primers if old
>> Check age of all DGGE reagents: acrylamide, formamide, APS, TEMED.
>> Are you using new reagents in PCR? Check if dNTPs, Taq, etc. are good.

BUFFER RE-USE
There is some discussion of how many runs can be done on a single tank of buffer before replacing the buffer is required. In my experience, there is a maximum of 4-5 runs per tank. The Ingeny system recommends that you replace 5L of buffer every run and replace the entire tank (17L) every 3 runs. I have had no problem running 4 gels without replacing the buffer at all on the Ingeny. It is advisable, however, to use fresh buffer if the gel is to be for a publication.

In addition, if you run your buffer at 0.5X TAE, it may be necessary to change buffer more frequently than with a 1.0X TAE buffer strength.

Detection issues / DGGE SENSITIVITY
· Li Zhang, Stephen Danon, Martin Grehan, Adrian Lee, and Hazel Mitchell. 2005. Template DNA Ratio can Affect Detection by Genus-Specific PCR–Denaturing Gradient Gel Electrophoresis of Bacteria Present at Low Abundance in Mixed Populations. Helicobacter. Volume 10 Issue 1 Page 80.
· Trulzsch B, Krohn K, Wonerow P, Paschke R. 1999. DGGE is more sensitive for the detection of somatic point mutations than direct sequencing. Biotechniques. 27(2):266-8.

DGGE ANALYSIS OF CLONES
In some cases, PCR conditions optimized for environmental samples do not work well for PCR amplification of clone DNA. In part, I suspect this is due to the exceedingly high concentration of DNA added to the PCR reaction from a clone (can be as cellular material or boiled cellular material or from plasmid extraction) and the high copy number of identical sequence. I recommend a higher dilution of DNA, fewer cycles, lower magnesium concentration, and higher annealing temperature – something to reduce this problem when amplifying clones. I have noticed that in general, pure culture DNA can behave differently in PCR reactions that in mixed cultures or environmental samples. This can make optimization of new primer sets difficult. One could spike environmental samples with target DNA and perform optimization thusly to avoid such a problem.

· S.A. Middleton, G. Anzenberger, and L.A. Knapp. 2004. Denaturing gradient gel electrophoresis (DGGE) screening of clones prior to sequencing. Molecular Ecology Notes 4 (4), 776-778.

Darek Bulinski has this to say about analyzing multiple clones per lane: “If we have a lot
to screen we run ten, 5 microliter samples per lane. This allows us to look at a large number of samples per DGGE gel. If any given lane contains band(s) that correspond to our whole community sample, we then run the samples in individual lanes on a second gel to identify the clone from which it was amplified. Also, for running clones on a DGGE gel, we pick a colony into 100 microliters of PCR water, vortex it and use 1 microliter per 25 microliter reaction (we've never had problems with this or required boiling). Then running 5 microliters of that on a DGGE gel is plenty (more if the amplification was less efficient).”

GC CLAMPS
Remember to put your GC clamp at the 5’ end of the primer!

· RM Myers, SG Fischer, T Maniatis and LS Lerman. 1985. Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. 13, 3111-3129.
· RM Myers, SG Fischer, LS Lerman, and T Maniatis. 1985. Nearly all single base substitutions in DNA fragments joined to a GC- clamp can be detected by denaturing gradient gel electrophoresis. Nucleic Acids Res. 13: 3131 - 3145.

I just happened across a website suggesting that having GC clamps on both PCR primers yielded better separation. I have had poor luck when I accidentally ordered GC clamps for both primers, but it appears this can vary according to the primer set. [See “Bipolar clamping versus monopolar clamping” on this website: http://www.charite.de/bioinf/tgge/ ]

HIGH DIVERSITY ENVIRONMENTS – MEASURING DIVERSITY
Many users have had problems performing DGGE analyses on high diversity environments such as soils. When applying general bacterial primers to the systems, with subsequent DGGE analysis, the gels can look smeared, in part due to the very high number of bands (many of the weak and indistinct). Molecular analyses such as DGGE, TGGE, TRFLP, SSCP can all be troublesome with such high diversity. In such cases, the best approach is probably to build a clone library from the PCR product and generate diversity estimates using rarefaction type analyses (see DOTUR, below). DGGE really reaches its limitations when dealing with such high diversity limitations. I would recommend to anyone dealing with such a high diversity to design narrower primers to apply to the system. Thus, one can focus on, for example, Actinomycetes, for which there are highly specific primers. You can use those primers directly for DGGE or nest them with general bacterial primers.

· DOTUR: http://www.plantpath.wisc.edu/fac/joh/DOTUR/documentation.html
· Schloss, P.D. & Handelsman, J. 2005. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Applied and Environmental Microbiology. 71(3):1501-1506.
· Huges JB, Hellmann JJ, Ricketts TH, Bohannan BJM. 2001. Counting the Uncoutable: Statistical Approaches to Estimating Microbial Diversity. Applied and Environmental Microbiology 67 (10) 4399-4406.
· Curtis TP, Sloan WT. 2004. Prokaryotic diversity and its limits: microbial community structure in nature and implications for microbial ecology. CURRENT OPINION IN MICROBIOLOGY 7 (3): 221-226
· William T. Sloan and Jack W. Scannell. 2002. Estimating prokaryotic diversity and its limits. PNAS 99 (16): 10494-10499.

MIGRATION OF BANDS INTO THE GEL IS LIMITED OR ABSENT? (NO VISIBLE BANDS IS A FREQUENT COMPLAINT)
Things to check:
>> Are voltage/amperage levels appropriate?
>> Are there bubbles underneath the gel (Ingeny)
>> Did you pour the gel correctly (i.e. put the high and low acrylamide solutions in the correct wells)?
>> Did you turn on the power supply (this just happened to somebody I know…!).
>> Are the electrode terminals clean (Bio-Rad)?
>> If you have only one gel, do you have a back glass plate to enclose the upper buffer reservoir?
>> Is there a leak in the upper buffer reservoir?
>> Make sure the lid is securely closed (Bio-Rad).
>> Is your stain fresh? GelStar, SybrGreen, etc. all are light sensitive and degrade.
>> Do you have a strong PCR product? Have you added enough DNA?
>> Did you electrophorese for too long (or too high a voltage) and the fragments migrated out of the gel?
>> Did you hook up the electrodes in the right order?
>> Did you let the product diffuse out of the wells in the gel?
>> Did recirculating buffer mix the PCR product in each well?

MULTIPLE BANDING FROM SINGLE POPULATIONS AND MULTIPLE SEQUENCES FROM SINGLE POPULATIONS

· Klappenbach JA, Dunbar JM, Schmidt TM. 2000. rRNA operon copy number reflects ecological strategies of bacteria. Appl. Environ. Microbiol. 66:1328-33.
· S.P. Gafan and D.A. Spratt. 2005. Denaturing gradient gel electrophoresis gel expansion (DGGEGE) - An attempt to resolve the limitations of co-migration in the DGGE of complex polymicrobial communities. FEMS Microbiol Lett. 2005
· A. Schmalenberger and C.C. Tebbe. 2003. Bacterial diversity in maize rhizospheres: conclusions on the use of genetic profiles based on PCR-amplified partial small subunit rRNA genes in ecological studies. Mol Ecol. 12(1):251-62.
· Nubel U., Engelen B., Felske A., Snaidr J., Wieshuber A., Amann R.I., Wolfgang L. and Backhaus H. 1996. Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J. Bacteriol. 178(19), 5636-5643.
· Satokari R.M., Vaughan E.E., Akkermans A.D.L., Saarela M., and de Vos W.M. 2001. Bifidobacterial diversity in human faeces detected by genus specific PCR and denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 67(2),504-513.
· Crosby, L. D., and C. S. Criddle, 2003. Understanding systematic error in microbial community analysis techniques as a result of ribosomal RNA (rrn) operon copy number. BioTechniques. 34(4), 790-803.

NON-RIBOSOMAL RNA GENE ANALYSES FOR BACTERIAL COMMUNITY ANALYSIS

· Dahllof, Baillie & Kjelleberg. 2000. rpoB based microbial communityanalysis avoids limitations inherent in 16S rRNA gene intraspeciesheterogeneity. Appl. Env. Micro. 66:3376-3380.

PCR ISSUES (There’s no end to these…)

Chimeras / Heteroduplex formation
Chimeras are always a concern in PCR analyses. To check to see if your sequence might be a chimera, you can use the chimera_check tool at the ribosomal database project (at least for ribosomal RNA gene sequences). You can also cut your sequence in half and BLAST each half of the sequence to see if you get similar results. There is also a new program called Pintail which is even more sophisticated.

http://www.cf.ac.uk/biosi/research/biosoft/Pintail/pintail.html
http://geta.life.uiuc.edu/RDP/misc/check_help.html
http://rdp8.cme.msu.edu/cgis/chimera.cgi?su=SSU

· Wang, G. C.-Y., and Y. Wang. 1997. Frequency of formation of chimeric molecules as a consequence of PCR coamplification of 16S rRNA genes from mixed bacterial genomes. Appl. Environ. Microbiol. 63:4645–4650.

“…Here evidence is presented for heteroduplexes as a major source of artifacts in mixed-template PCR…Heteroduplexes became increasingly prevalent as primers became limiting and/or template diversity was increased. … the diversity of artifactual sequences increases exponentially with the number of both variable nucleotides and of original sequence variants. Our model illustrates how minimization of heteroduplex molecules before cloning may reduce artificial genetic diversity detected during sequence analysis by clone screening. Thus, we developed a method to eliminate heteroduplexes from mixed-template PCR products by subjecting them to ‘reconditioning PCR’, a low cycle number re-amplification of a 10-fold diluted mixed-template PCR product. This simple modification to the protocol may ensure that sequence richness encountered in clone libraries more closely reflects genetic diversity in the original sample.”

· Janelle R. Thompson, Luisa A. Marcelino and Martin F. Polz. 2002. Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucleic Acids Research. 30(9): 2083-2088.

Degenerate primers
Some functional gene (i.e. non ribosomal RNA gene) primers are highly degenerate and this can cause problems during DGGE analyses. Since the whole point of DGGE is to separate fragments that differ in sequence, identical PCR fragments that have different primer sequences can sometimes generate multiple bands on DGGE. The problem originates during the PCR reaction. When using degenerate primers, a low annealing temperature must be used to accommodate all the possible primer combinations (there is no point in having degenerate primers and then using an annealing temperature too high for some of the primers). However, at the low annealing temperature, some of the primers can anneal non-stringently to DNA target, and thus the non-stringent primer becomes incorporated into the growing DNA fragment. So, multiple primer combinations can anneal to the same template DNA and generate copies of the same fragment, but with different primer sequences. If these primer sequences are great enough, DGGE analysis will separate out the identical PCR fragments by the differences existing in the primer region. Sequencing/clone DGGE analysis can help resolve this issue. However, there isn’t much that can be done to avoid this problem – and it may again complicate measurements of diversity or dendogram analysis of DGGE gels. As a side note, this is the reason that you should NEVER submit the primer region of your sequence to Genbank.

Double bands
This appears to be another major complaint in the DGGE literature. Since many of the issues with double banding are a result of the PCR stage, you may want to consider:
>> Longer final elongation time (5-30 minutes of 72C)
>> Slow touchdown to 4C after final elongation stage
>> Check primer degeneracy
>> Concentration of DNA added to reaction
>> number of PCR cycles.

· Janse I, Bok J, Zwart G .2004. A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis. Journal of microbiological methods. 57:279-281.

PCR Inhibition due to humics and polysaccharides
DGGE analyses can often be limited by the PCR step of the process. The PCR step can, in turn, be limited by the quality of the DNA extraction. Environmental samples rich in humic acids (organic rich soils, composts, decaying litter, etc.) and polysaccharides (biofilms, cyanobacteria, microbial mats, etc.) can contribute to poor quality DNA extracts. A lot of effort has been expended to deal with such environmental contaminants. In general, phenol/chloroform extractions and ultracentrifugation in a Cesium chloride gradient are the most effective for recovering pure DNA, these methods can be tiresome and contain toxic chemicals.

For humic acids, MoBio has a very nice kit for extracting soil DNA: The MoBio “PowerSoil” DNA isolation kit. I have also noticed that you can reduce the amount of humic acids that you recover in your extracts by removing EDTA from the extraction buffer, and by repeated cleaning of the DNA with guanidine thiocyanate, or by cleanup on PVPP columns. For a reference, see the following article; I would be happy to provide a more detailed protocol if necessary. I have read that “T4Gene” 32 protein can be added to PCR reactions to reduce humic inhibition.
· Inbar, E., Green, S.J., Hadar, Y. and D. Minz. 2005. Competing Factors of Compost Concentration and Proximity to Root Affect the Distribution of Streptomycetes. Microbial Ecology. 50:73-81.
· LaMontagne MG, Michel FC Jr, Holden PA, Reddy CA. 2002. Evaluation of extraction and purification methods for obtaining PCR-amplifiable DNA from compost for microbial community analysis. J Microbiol Methods. 2002 May;49(3):255-64.
· CC Tebbe and W Vahjen. 1993. Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and a yeast. Appl. Environ. Microbiol., 59(8): 2657-2665.

For polysaccharides, I recommend a potassium ethyl xanthogenate method.
· Tillett, D. and Neilan, B.A. 2000. Xanthogenate nucleic acid isolation from cultured and environmental cyanobacteria. J. Phycol. 36:251-258.

You can also try to overcome PCR inhibition due to contaminants by application of a “pre-PCR” stage using a specialized polymerase to make a large number of copies of genomic DNA.

· Gonzalez et al. 2005. Multiple displacement amplification as a pre-polymerase chain reaction (pre-PCR) to process difficult to amplify samples and low copy number sequences from natural environments. Environmental Microbiology. 7(7):1024-1028.

PCR Contamination
PCR contamination is a common event in molecular labs. This is particularly true for general bacterial PCR primers which will amplify any bacterial DNA that could be floating around in your lab. The first step to dealing with such contamination is to throw away all your PCR reagents (if this isn't too painful). I tend to use aliquots of each reagent and after having opened a tube I either dispose of the tube or use it for a less contamination likely PCR reaction (i.e. with a specific gene primer). You should think about where the contamination could come from. If one of your stock solutions (say primer stock) has been contaminated, you're in trouble. You can try a little test in somebody else's lab and PCR each of your reagents. You may also have some sort of contamination on your pipettes. Some researchers also use filters to clean up contamination - this is a last resort effort. To do this, you can filter all your "master mix" for PCR (WITHOUT THE ENZYME) through a 30 KD filter. The filter will pass the PCR primers, but will not pass the polymerase or genomic DNA (thus, add the enzyme afterwards). You can also work with filter tips that are not autoclaved but are ordered DNA/RNAse free; likewise with PCR tubes. Use purchased DNAse free PCR water. If none of that works, find a new career....

· Meier et al. 1993. Elimination of Contaminating DNA within Polymerase Chain Reaction Reagents: Implications for a General Approach to Detection of Uncultured Pathogens. Journal of Clinical Microbiology, 31:646-652.

Primer cleanup
When ordering primers I have never used anything but the most standard cleaning (i.e. desalting) offered by the companies. I have never found any improvement in DGGE analyses with reverse-phase HPLC, PAGE, or reverse-phase cartridge (RP1). Others may have different experience. Dr. A.G.C.L. (Arjen) Speksnijder reports that they have had poor experience with HPLC cleanup.

Primer Mistmatches / PCR Bias

· Kousuke Ishii and Manabu Fukui. 2001. Optimization of Annealing Temperature To Reduce Bias Caused by a Primer Mismatch in Multitemplate PCR. Appl. Environ. Microbiol. 67(8): 3753–3755.
· Shinya Kurata, Takahiro Kanagawa, Yukio Magariyama, Kyoko Takatsu, Kazutaka Yamada, Toyokazu Yokomaku, and Yoichi Kamagata. 2004. Reevaluation and Reduction of a PCR Bias Caused by Reannealing of Templates. Appl. Environ. Microbiol. 70(12):7545-9.


Primer Design
· Melt Profile Program: http://web.mit.edu/osp/www/melt.html
· Primernet (http://www.primernet.com/)
· Primrose : KE Ashelford, AJ Weightman & JC Fry. 2002. PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. Nucleic Acids Research, Vol. 30, No. 15 3481-3489
o http://www.cardiff.ac.uk/biosi/research/biosoft/Primrose/primrose.html
· Probe Library (ARB): http://www.arb-home.de/

Quantification
· Park JW, Crowley DE. 2005. Normalization of soil DNA extraction for accurate quantification of target genes by real-time PCR and DGGE. Biotechniques. 38(4):579-86.
· Bruggemann J, Stephen JR, Chang YJ, Macnaughton SJ, Kowalchuk GA, Kline E, White DC. 2000. Competitive PCR-DGGE analysis of bacterial mixtures: an internal standard and an appraisal of template enumeration accuracy. J Microbiol Methods. 40(2):111-23.
· D.G. Petersen and I. Dahllöf. 2005. Improvements for comparative analysis of changes in diversity of microbial communities using internal standards in PCR-DGGE. FEMS Ecology. [In Press].

Optimization
· Wood GS, Uluer AZ.1999. Polymerase chain reaction/denaturing gradient gel electrophoresis (PCR/DGGE): sensitivity, band pattern analysis, and methodologic optimization. Am J Dermatopathol. 21(6):547-51.
· Markus M. Moeseneder, Jesús M. Arrieta, Gerard Muyzer, Christian Winter, and Gerhard J. Herndl. 1999. Optimization of Terminal-Restriction Fragment Length Polymorphism Analysis for Complex Marine Bacterioplankton Communities and Comparison with Denaturing Gradient Gel Electrophoresis. Applied and Environmental Microbiology, 65(8): 3518-3525.
· Vanessa M. Hayes, Ying Wu, Jan Osinga, Inge M. Mulder, Pieter van der Vlies, Peter Elfferich, Charles H. C. M. Buys, Robert M. W. Hofstra. 1999. Improvements in gel composition and electrophoretic conditions for broad-range mutation analysis by denaturing gradient gel electrophoresis. Nucleic Acids Research. 27(20): 29.

General PCR articles
· Speksnijder AG. 2001. Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences Appl Environ Microbiol. 67(1):469-72

PERPENDICULAR GELS
Optimization of the gradient for DGGE analyses can be done empirically by running an initial gel with a 0-80% gradient and then narrowing the gradient in subsequent analyses by inspection of the first gel. Alternatively, you can pour a perpendicular gel. In this case, during the casting stage the gel is turned on its side. After the gel has polymerized, the gel is set upright, and a single sample is analyzed. This can help identify which gradient, or if any gradient, will separate the fragments of interest. Protocols for pouring these gels come with the Bio-Rad system, but I don’t think the Ingeny system comes with the capacity to pour such a gel.

POLYMERIZATION
Troubleshooting DGGE gels that do not look good can include problems with the pouring and polymerization of your acrylamide. In addition to checking that your reagents are fresh, you may want to look at the following website:
· http://www.bio-rad.com/LifeScience/pdf/Bulletin_1156.pdf

You may also want to add commercially available substances to your acrylamide to enhance the strength of your gel and reduce tearing. Some users have complained that this, or silanizing glass plates made DGGEs worse. Oscar J. de Vos from Wageningen University indicates that UV light cannot pass through the Gelbond product listed below. Still, you may want to check this out:
http://www.cambrex.com/Content/bioscience/CatNav.oid.520.prodoid.GelbondPAG

There has been some discussion in the DGGE group about how long you should let your gels polymerize. In general, 1.5 hr seems to be the absolute minimum. You should also not move your gel during polymerization, if possible. Some users have suggested that 2 hr polymerization is adequate and should not be longer than this. I have not found that length of polymerization time before running is particularly important, provided the 1.5 hr time is reached. If storing gels, I wouldn’t recommend more than 1 or 2 days. We used to put the gels in a plastic bag with a moist kimwipe when storing overnight. I have left gels out of the refrigerator overnight without any detrimental effect. You should make sure that there is some moist towel or kimwipe to ensure that the gel does not dry out.

SEPARATION OF BANDS
If you are not getting good separation on your DGGE, there can be a number of issues to examine. First, not all genes and regions of genes are suited to DGGE analyses. There is a rule of thumb which suggests that if DNA fragments do not differ by 1% or more (is it true?), it will be difficult to resolve them by DGGE. You can reduce the size of the PCR fragment, and this may increase separation if you don’t eliminate variable sites. You should check, if possible, available sequences to see if there are reasonable differences in sequence that would allow separation. Also, remember that in general, large fragments (>600 bp) do not separate well by DGGE. There is the 1650 bp fragment of fungal 18S ribosomal RNA, but this appears to be a freakish exception.
· E.J. VAINIO and J. HANTULA. 2000. Direct analysis of wood-inhabiting fungi using denaturing gradient gel electrophoresis of amplified ribosomal DNA. Mycological Research 104: 927-936.
>> An improper gradient can yield poor DGGE results. If the gradient is too wide, band may migrate very closely and you will loose reasonable separation; however, these gels tend to have the sharpest bands and look very nice. One should be careful not to make too narrow a gradient as this can yield fuzzy bands.
>> Check to see that the voltage and run time are adequate.
>> Check to see that there are no blockages to current running through the system (check that the amperage is appropriate for your system).
>> Did you remember to put a GC clamp on one of the primers?
>> Did you put a GC clamp on both primers? If so, whoops…
>> Do you have more than one sequence in your system? If not, perhaps you should be getting a single band…

· Y Wu, VM Hayes, J Osinga, IM Mulder, MW Looman, CH Buys, and RM Hofstra. 1998. Improvement of fragment and primer selection for mutation detection by denaturing gradient gel electrophoresis. Nucleic Acids Research. 26:5432-5440.
· Kisand V, Wikner J. 2003. Limited resolution of 16S rDNA DGGE caused by melting properties and closely related DNA sequences. J Microbiol Methods. 54(2):183-91.

SINGLE STRANDED DNA
Single stranded DNA can sometimes cause problems with DGGE. If this is your particular problem, you can try digesting it away prior to loading your PCR product on DGGE using Mung Bean nuclease.

SMILING GELS
Smiling of bands near the edges of DGGE gels appears to be endemic in all systems. While the exact cause of this is not entirely clear, the smiling effect can be held in check by two approaches, best used together:
· Don’t load PCR product in the very far lanes.
· Apply grease to the spacers. Older spacers may require more grease than new spacers, but don’t overload the spacers with grease. A thin film is adequate.

· Brinkhoff, T. and Van Hannen, E.J. 2001. Use of Silicone Grease to Avoid 'smiling Effect' in DGGE. Journal of Rapid Methods and Automation in Microbiology 9:259-261.

STORING GELS AFTER ELECTROPHORESIS
According to James Hollibaugh, DGGE gels can be stored for subsequent extraction of DNA by drying them onto filter paper using a gel dryer. Once dry they can be stored at room temperature in a loose-leaf binder (use sheet protectors) for at least a year.
· Hollibaugh, J. T., P. S. Wong, N. Bano, S. K. Pak, E. M. Prager and C. Orrego. 2001. Stratification of microbial assembledges in Mono Lake, California, and response to a mixing event. Hydrobiologia 466:45-60.

WELLS – WAVY, POOR QUALITY, ETC.
Sometimes the wells that are formed when you remove the comb are of poor quality, usually because the polymerized acrylamide that forms the walls between the wells does not stand up straight and collapses. You can rectify this after the fact by using a syringe tip or other similar device to manually straighten each well.

To avoid this problem in general, I have found that one of the causes of this is to pour TOO MUCH acrylamide into the comb area (meaning that you over-pour the amount of acrylamide in the stacking gel area). For whatever reason, if you are careful to add just the amount needed, you’ll have less problems. Remember not to add too little, because acrylamide shrinks as it polymerizes. Putting plastic wrap over the top of the gel has been suggested to reduce evaporation and limit shrinkage.

Also, pull your comb out very slowly.

DGGE OF HIGH GC PCR FRAGMENTS
· Per Guldberg1, Kirsten Grønbæk, Anni Aggerholm, Anton Platz, Per thor Straten, Vibeke Ahrenkiel, Peter Hokland and Jesper Zeuthen. 1998. Detection of mutations in GC-rich DNA by bisulphite denaturing gradient gel electrophoresis. Nucleic Acids Research. 26(6): 1548-1549.
· Ying Wu, Rein P. Stulp, Peter Elfferich, Jan Osinga, Charles H. C. M. Buys, Robert M. W. Hofstra. 1999. Improved mutation detection in GC-rich DNA fragments by combined DGGE and CDGE. . Nucleic Acids Research. 27(15): 9.


FANCY DGGE TECHNIQUES
· Van Orsouw NJ, Vijg J. 1999. Design and application of 2-D DGGE-based gene mutational scanning tests. Genet Anal. 14(5-6):205-13.
· Nathalie J. van Orsouw, Rahul K. Dhanda, R. David Rines, Wendy M. Smith1, Iakovos Sigalas, Charis Eng1, Jan Vijg. 1998. Rapid design of denaturing gradient-based two-dimensional electrophoretic gene mutational scanning tests. Nucleic Acids Research. 26(10): 2398-2406.
· Green, S.J. and D. Minz. 2005. Suicide Polymerase Endonuclease Restriction (SuPER) – a novel technique for enhancing PCR amplification of minor DNA templates. Appl. Environ. Microbiol. 71:4721-4727.
· Cremonesi L, Firpo S, Ferrari M, Righetti PG, Gelfi C. 1997. Double-gradient DGGE for optimized detection of DNA point mutations. Biotechniques. 22(2):326-30.
· Burmeister, M., diSibio, G., Cox, D. R., and Myers, R. M. Identification of polymorphisms by genomic denaturing gradient gel electrophoresis: application to the proximal region of human chromosome 21. Nucleic Acids Res 19(7): 1475–81, 1991.
· William E. Holben, Kevin P. Feris, Anu Kettunen, and Juha H. A. Apajalahti. 2004. GC Fractionation Enhances Microbial Community Diversity Assessment and Detection of Minority Populations of Bacteria by Denaturing Gradient Gel Electrophoresis. Appl Environ Microbiol. 70(4): 2263–2270.

RECOMMENDED PCR-DGGE PRIMER SETS AND GRADIENT CONDITIONS

Bacterial 16S rRNA gene: 341F / 907R (E. coli numbering. See Muyzer References; Primer in bold):

341F-GC: 5’-CGC CCG CCG CGC CCC GCG CCC GTC CCG CCG CCC CCG CCC GCC TAC GGG AGG CAG CAG-3’
907R: CCG TCA ATT CMT TTG AGT TT

PCR conditions: 4.0 mM Mg; 60C Annealing temperature
Nest-PCR conditions: 4.0 mM Mg; 64C Annealing temperature; 28 cycles
DGGE conditions: 25/30 to 60/70% denaturant; 100V; 60C; 17 hr; 6% acrylamide


Keywords: denaturing, gradient, gel electrophoresis, help, advice, guide, DGGE, community composition, molecular biology, fingerprinting, PCR-DGGE, nested PCR.

155 Comments:

At 11:25 PM, Anonymous Anonymous said...

I must say! tremendous amounts of effort put in explaining DGGE... Great work... I am sure many will use this as a guide. You will be famous one day.

 
At 1:14 AM, Anonymous Anonymous said...

Thank you so much for a clear and thorough DGGE protocol!! :)

 
At 6:35 AM, Anonymous Anonymous said...

Absolutely brilliant!
:D

 
At 12:32 PM, Anonymous Anonymous said...

Fantastic blogg! I find it extremely useful, thanks so much for doing this!!

 
At 3:57 PM, Blogger Dexter said...

Thank you guys very much for building up this DGGE technology specific website. It's very great because I found a lot information need for my research!

 
At 7:20 AM, Blogger Rok said...

Good job! Well done! I will recommend your blog to my colleagues and students.

Rok

 
At 1:39 AM, Anonymous Anonymous said...

Not sure what you mean-

'..what is the point? I would like to suggest that a better way to approach this is to clone directly from the original PCR product (using GC-clamped PCR primers) and then screen the clones against the environmental sample...'

Can somebody elaborate?

 
At 10:59 AM, Anonymous Anonymous said...

Thank you very much.

This blog is fantastic. It was very useful for me.

Congratulations and I hope that you continue writting about this topics.

 
At 11:30 PM, Anonymous Anonymous said...

Hi Stefan,

I`d like to thank you first for the great jib!
I got a Q, At first I have no bubbles trapped but after a few hours having it run, bubbles appear and get stuck in the middle of the gel!!! What`s wrong?

I appreciate it in advance.
(Plz email me your reply if possible at sam_phd2006@yahoo.com)


Cheers,

Sam

 
At 8:50 PM, Blogger someone said...

i have a question about DGGE. can anyone answer me , is formamide neccessary for DGGE?

 
At 8:14 PM, Anonymous Anonymous said...

does anyone know how to best clean plates after using silicone grease on the spacers? is there a particular solvent that works for this? so far i've only succeeded in smearing the stuff all over the plates.

 
At 3:37 AM, Anonymous Anonymous said...

A very good guide. Thanks.

 
At 10:10 AM, Anonymous Anonymous said...

Is there anyone that can help me with post DGGE? I've excised the bands from the DGGE gel, and washed with ddwater and then eluted overnight with elution buffer. I then used the top layer as template DNA in a PCR with the same DGGE primers but without the GC clamp. Im getting double bands on the agarose gel. wat could be the reason for this and how can I eliminate it?

Thanks alot!

 
At 1:00 PM, Blogger Stefan J. Green said...

In response to the most recent comment - I would suggest that you try to raise the annealing temperature of your post-DGGE amplification, or dilute the DNA template (try a range of dilutions), or decrease the number of PCR cycle. You can often get spurious bands when working with very high concentrations of DNA.
Let me know if that helps,
Stefan

 
At 12:28 PM, Anonymous Anonymous said...

Awesome site! thank you so much for making this information so readily available and simple to understand!!!

 
At 11:56 PM, Anonymous multiple band ligator manufacturer said...

Such a great post.......

 
At 12:01 PM, Anonymous Anonymous said...

Nice work and it is VERY helpful! I will recommend it to my colleagues.

 
At 7:42 PM, Anonymous Panic Attack said...

This DGGE technology specific is a great aid to research. It must have took a lot of time and effort on your part to deliver to us such a detailed and comprehensive report. I appreciate the clarity of presentation of the DGGE protocol.

 
At 2:49 AM, Anonymous ANU said...

VERY INFORMATIVE AND THANKS .......EXPLANATION IS VERY CLEAR...NICE TIPS TOO...

 
At 2:31 AM, Anonymous puertas metalicas said...

So, I don't actually believe this will work.

 
At 4:33 AM, Anonymous Rahul said...

Hii Stefan,
Thanks for the guide its really helpful. I have just started with DGGE and was finding it tough to optimize untill I read this. Currently I am able to amplify community DNA with GC clamps and make a decent gradient gel as well. The only recurring problem is I am not to make proper wells (lack of polymerization due to oxygen presence I guess). I use 0.09% (25 ul) each of TEMED and 10% APS for a 20 ml gel as directed in BioRad manual. Have tried with higher volumes as well. 100 ul APS and 50 ul TEMED doesnt give me sufficient time to cast. Please help me save precious resources and time in optimization.

Thanks.

 
At 11:42 AM, Anonymous Anonymous said...

Dear Stefan,
If, I should compare different soils, (comparing different fingerprinting or asses the different diversity index) how much DNA I should add to pcr reaction? Should I measure, with the spectrophotometer, the dna amount of each sample and add the same amount in each pcr? Or Should I take the same amount of soil (5 g for instance) perform the dna extraction and purification and add directly 2 (for instance) microliter of dna in each pcr?"

 
At 1:11 AM, Anonymous Rt Pcr Primer said...

Very nice article.The post was consistent and clear.I'll look forward to it and would like to share it also.

 
At 11:09 AM, Anonymous Anonymous said...

Black Jimmy Graham Women's Jersey jouccanda
Arian Foster Elite Jersey jouccanda
Sean Taylor womens Jersey jouccanda
http://www.nikecardinalsnflstore.com

 
At 3:06 PM, Anonymous Anonymous said...

Doug Martin Orange Jersey axiotakix
Black Darren McFadden Mens Jersey axiotakix
Jimmy Graham Women's Jersey axiotakix
http://www.nikeseahawksnflstore.com

 
At 4:46 PM, Anonymous Anonymous said...

Larry Fitzgerald Cardinals Jersey axiotakix
wholesale jerseys china axiotakix
Doug Martin Youth Jersey axiotakix
http://www.nikeredskinsnflstore.com

 
At 7:31 PM, Anonymous Anonymous said...

Green Aaron Rodgers Women's Jersey axiotakix
Women's Drew Brees Jersey axiotakix
Peyton Manning Game Jersey axiotakix
http://www.nikecardinalsnflstore.com

 
At 9:26 AM, Anonymous Anonymous said...

Russell Wilson White Jersey
Sidney Rice Blue Jersey
James Harrison Womens Jersey
drydayoutraro

 
At 8:09 PM, Anonymous Anonymous said...

Larry Foote Womens Jersey
Golden Tate White Jersey
Isaac Redman Youth Jersey
drydayoutraro

 
At 2:54 AM, Anonymous Anonymous said...

axotomarvex Adam Vinatieri Jersey
T.Y. Hilton Jersey
Andrew Luck Jersey
UnmannaSmurce

 
At 7:48 AM, Anonymous Anonymous said...

Calvin Johnson Jersey
Golden Tate White Jersey
Bobby Wagner Women's Jersey
drydayoutraro

 
At 2:48 PM, Anonymous Anonymous said...

Biansioni www.nikegiantsnflshop.com Clay Matthews Jersey Antonio Brown Youth Jersey

 
At 2:48 PM, Anonymous Anonymous said...

Jerricho Cotchery Jersey
Jonathan Dwyer Youth Jersey
Earl Thomas Grey Jersey
drydayoutraro

 
At 5:33 PM, Anonymous Anonymous said...

Helo ! Forex - Работа на дому на компьютере чашка кофе получать удовольствие от работы получать деньги, просто зарегистрируйтесь forex [url=http://foxfox.ifxworld.com/]forex[/url]

 
At 7:55 PM, Anonymous Anonymous said...

Uncut spell, a construction companionship turned up to start erection a building on the tire out of the closet lot.

The 484678 [url=http://mios.my-board.org/sdh.html]450216[/url] [url=http://mios.my-board.org/sdu.html]824430[/url] 175015 [url=http://poa7.000space.com/yfd.html]567194[/url] green family's 5-year-old daughter as expected took an order forth in all the

troops flourishing on next door and drained much of each lifetime observing the workers.

 
At 4:04 AM, Anonymous Anonymous said...

Hey this is kind of of off topic but I was wanting
to know if blogs use WYSIWYG editors or if
you have to manually code with HTML. I'm starting a blog soon but have no coding skills so I wanted to get guidance from someone with experience. Any help would be enormously appreciated!
Feel free to visit my homepage : work from Home jobs

 
At 8:35 AM, Anonymous Anonymous said...

chwilowka przez internet bez bik i krd
pożyczki pozabankowe
pożyczki pozabankowe
kredyt bez bik
pożyczka 40000 zł
pożyczki pozabankowe 0 prowizji
o nas

 
At 7:13 AM, Anonymous Anonymous said...

With only few clicks[url=http://www.officialblackhawksauthentic.com/jonathan-toews-authentic-jersey.html]Jonathan Toews Authentic Jersey[/url]
you could get everything done and simply wait for the items on your doorstep Good Discount Nfl Jerseys From ChinaDo you know someone that does not aware about the strength of China and the great progress they made in the modern age? I can assure you that everyone aware about it
You may also outsource the job to another person in order toThere are various areas around the net had been one particular can locate psp movie downloads and psp songs downloads

 
At 9:57 PM, Anonymous Anonymous said...

I am using Ingeny for DGGE. Is it advisable to run DGGE for 2 specific primer (with different acrylamide gel percentage) at the same time (front and back gel)? Thank you. I wish to excise the band.

 
At 9:05 PM, Anonymous Anonymous said...

Well worded inquiries will reveal the most information about a candidate5- Life is about understanding Old habits are hard to break! Habits are especially hard to break when there is no support for the new skills and behaviors back in the workplace Don't keep wires where your puppy may gnaw or chew on them
The native Texan resides in Plano, Texas, a heartbeat north of Dallas, and doesn't wear a 10-gallon hat or cowboy bootsWith webmail set up, all you have to worry about is remembering your email login and password These are changes you can make that will change the way your dog thinks about you If someone you love is dying, be grateful for the time you had with them

Arian Foster Blue Jersey
Peyton Manning Youth Jersey

Thought is like hammering a nail Give away what you want more of ? it really worksFree Will, in my view, is the way we perceive our life as well as the way we portray ourselves We using graphology chooses right employees according to business criteria

Arian Foster Women's Jersey

 
At 3:45 PM, Anonymous Anonymous said...

The one thing that woodland gardens require, however, is patience. [url=http://www.mulberryhandbagssale.co.uk]Mulberry ooutlet online[/url] I have done the research and reading up on my topic, but the thought of writing it down scares the hell out of me. [url=http://www.goosecoatsale.ca]canada goose expedition[/url] Qzrpltljw
[url=http://www.pandorajewelryvip.co.uk]pandora store[/url] Uvsteutmg [url=http://www.officialcanadagooseparkae.com]canada goose coats[/url] jcwvmvbav

 
At 5:53 PM, Anonymous Anonymous said...

I have been using

a Chi straightener for most of my life and I thought it was the best. This past Christmas I bought this straightener for my mom. I used it all month and the difference is amazing. I don't know if it's

because of the titanium plates or the degree to which it heats, but it straightens the hair in one pass through and leaves it SO SHINY. My hair stays straight longer after using this, too. I love it and have

made the switch to babyliss!

 
At 4:53 PM, Anonymous Anonymous said...

This item is great for people w curly unruly hair. I have fine curly hair, just a lot of it - so sometimes products for curly thick (the folicle itself) do not give achieve the

results I want for my hair. The great thing is you only need a couple of pumps (too much can make your hair kind of hard - as if you were using a gel) and give you super defined curls w/o using gels,

hairsprays, individually twisting your curls, etc... It just gives you what you hope nature intended your hair to look like, w/o looking like you have a product in your hair - It has a nice mild smell and

gives you soft and bouncy curls - I use it to style and comb my hair (when wet) and then finish with a bit of MoroccanOil and have hair that looks great all day long

 
At 3:16 AM, Anonymous Anonymous said...

This is the best thing ever!! I live in FLorida where it is hot and humid....I have no air

conditioning in my car so I have to drive with the windows down...I have very curly, thick, and out of control hair...with all these elements it is hard to find something to make my hair stay straight. This

worked...I drove on the highway with my windows down in nice hot humid weather and it is still as perfect as when I straightened it 2 days ago!! Buy this product it is worth the money!!!

 
At 12:42 AM, Anonymous Anonymous said...

Keep on ωrіting, gгеat ϳоb!


Herе is my pagе - Payday Loans Online Lenders
Feel free to visit my blog Payday Loans Online

 
At 6:24 PM, Anonymous Anonymous said...

top [url=http://www.c-online-casino.co.uk/]online casinos[/url] check the latest [url=http://www.casinolasvegass.com/]online casino[/url] unshackled no set aside hand-out at the best [url=http://www.baywatchcasino.com/]online casinos
[/url].

 
At 4:37 AM, Anonymous Anonymous said...

[url=http://saclongchampa.deviantart.com/journal/]longchamp soldes[/url] You've probably seen many of the different kinds of pet carriers on the market for people with small pets. Perhaps the most popular are the purse-style carriers. These come in all shapes, sizes, Save up 60% when shopping Mulberry Brynmore For Macbook Pro Leather Messenger Bag Light Coffee for Men at our Mulberry outlet. materials, and price tags. As its name implies, a laptop bag is a bag for your laptop. It is well-cushioned to make sure that your laptop and all its parts are intact even with vigorous movements or vibrations. Also, it has different compartments to store your laptop and its accessories (battery, cord, charger).
[url=http://longchamppaschers.blogmonster.de/]longchamp soldes[/url] Many of us do not have much budget from work to obtain the stuff we want, and have to get it ourselves, others have a great budget and are looking for ideas. Use Shop the latestMulberry handbags from the most popular stores, Sale Authentic Mulberry Zip Natural Leather Purse Light Coffee for Women at auction Tangle Therapy for hand and mind wellness. This Tangle is covered with soft, flexible texturized plastic.
[url=http://longchamppliagea.blog.petitmallblog.jp/]sac longchamp moins cher[/url] An honest seller will stand behind their products authenticity. Otherwise they would quickly be out of the business of selling famous designer name brand clothing. Also only buy from US sellers, have 100% feedback and have over 100 comments from buyers and strictly deal in designer clothing.. My partner and i run into a son it can be practically nothing will likely be on the lookout for talk about and just how all pages and posts would likely for the actual computer keys? A good: Virtually no should you not spine a wide factor can be better than a professional DIGITAL SLR. You have to have build. Your ipad 3gs (for accessibility)? A good: Certainly the actual Excellent Mulberry Outlet Medium Alexa Leather Satchel Camel Bag popular sale Mulberry Handbags UK Online effects of Fruit members like bristles inside full aniline-dyed Italian language synthetic leather...

 
At 11:56 PM, Anonymous Anonymous said...

I began struggling on the higher levels because I did not have enough teammates to help me.
A short video that requires minimal digital effects can be rendered in just a few minutes while a feature length animated movie
can take up to several weeks because of the complexity of the effects needed and the
3D animation itself. You just read the instruction, know which buttons to press to handle the vehicle and just jump into the game.


Check out my page; http://haduqoynhni.fotopages.com/?entry=6812003

 
At 2:50 PM, Anonymous Anonymous said...

Please supplement charge before use when the battery has been kept for a long
time. Remove other nearby wireless devices, such as a cordless
or cellular telephone and make sure the mouse is
at least eight inches away from a wireless keyboard.
Both Intel and AMD contain information built in the processor to allow the processor to be used efficiently with mobile computing saving
on battery life while giving the best performance.



Also visit my web site: myvideo downloader

 
At 10:35 PM, Anonymous Anonymous said...

Yοu can alѕo buy bundle deals, which most
raԁio statіons offer, to decreasе
the overall ad сοst. Μany people will be happy with replaceable batteries for home use and occasional outings.
These include thе artist-branded music chаnnels such as The Gratеful Dead Channel,
Eminеm's Shade 45, Jimmy Buffett'ѕ Radіo Maгgaritаville; there is evеn the
classical music Metropolitan Οpеrа Radio
channel.

Feel free to surf to mу web-sіtе radio sender

 
At 4:44 AM, Anonymous Anonymous said...

Tomb Raider, Mega Moolah, Mermaid's Millions, and Blackjack are some of the new Android game apps that have been released from Microgaming's partner, Spin 3.
If you want to enjoy the game thoroughly then
you need to get on board of a reliable online bingo
site to get the maximum enjoyment as well as benefit of the game.
Even when things seem hopeless, Kiko's narration provides some great comedy relief.

my blog post; radio sender

 
At 3:53 AM, Anonymous Anonymous said...

Completely I share your opinion. In it something is also to me this idea is pleasant, I completely with you agree.
Interestingly, and the analogue is?
Bravo, what phrase..., a magnificent idea
Let's talk.
Would like to tell to steam of words.

[url=http://spaces.covers.com/blog/cheapbag2][b]michael kors outlet online[/b][/url]
[url=http://cheapmkbag2.snappages.com/blog/2013/01/21/cheapmkbag2][b]michael kors outlet online[/b][/url]
[url=http://michaelkorsoutlet0.exteen.com/20130114/cheap-sale-mulberry-messenger-bags][b]michael kors outlet online[/b][/url]
[url=http://www.mvpmichaelkors.sitew.us/#Page_1.A][b]michael kors outlet online[/b][/url]
[url=http://shenenmaoyiqwe.angelfire.com/][b]michael kors outlet online[/b][/url]

 
At 2:59 PM, Anonymous Anonymous said...

Hi there, after reading this amazing post i am too cheerful to
share my experience here with colleagues.

Feel free to surf to my webpage :: tv shows

 
At 5:09 PM, Anonymous Anonymous said...

w$p1erajmy hosp1cja Zaraźliwy Rzeczywistość Muzyka Łańcuch Słuchać Spodnie Make-up II Energia Sink I Dach Rosnąć Koncert Gleba II Grosz Historia Kwaśny Mąka Rzucać Przód Dolina Bezwzględny Minuta Opinia Powłoka Sektor w$p1erajmy hosp1cja Harmonogram Finansowy Przyjaźń Propozycja Zrobić Wentylator Rynek Opinia Artysta Ciotka Zdrowy Wyborczy Lok Poza Wzrok Owoc Szczątki Wyścigi Paszport Zupa Warzywo Dach Noc Rosnąć Naklejać [url=http://di.pl/user/13136-earn4me/]w$p1erajmy hosp1cja[/url] Tabletka Przeciwnik Rana Okazja Nienawidzić Lód Kwas Swallow i Rzucać Zwierzę Humor Otwarte Maksymalny Cable II Netto Otwarcie Basen Wyobraźnia Restauracja Słyszeć Humorystyczny Osobowość Miś II Koniec Cały [url=http://wiki.goldencup.ir/index.php?title=Geneva]w$p1erajmy hosp1cja[/url] Medycyna Pauza Komputer Święty Rok Ludzki Przepraszam Dopasować Dorosły Nacisk Babcia Obietnica Drewno Włosy Danie Miąższ Uczyć się Wypadek Ankieta I Pamiętnik Podniesienie Kontynent Wskazać Pogawędzić Pies [url=http://start.uni-guehlen.de/w/index.php?title=Inesita]w$p1erajmy hosp1cja[/url] Cel Produkować Opon Szuflada II Wąski Śmieci Poemat Groth Tajemnica Rewizja Atrakcyjny Równoważny Kosztować Hide II Gorączka Minimum Prawny Język Naprawiać Kojarzyć Blind II Kandydat Ciągnąć Magazine II Obudowa [url=http://www.zinzipedia.de/wiki/index.php?title=Malinda]w$p1erajmy hosp1cja[/url] Schronisko Różny Piłka nożna Sprzęt Pytanie Szuflada II Płatność Tachnique Entuzjastyczny Potajemnie Owoc Marketing Wspiąć się Kino Cable II Symbol Szczotka III Organizacja Zilustrować Rozmowa Z wyjątkiem Kolarstwo Nagradzać Spokojnie Uczucie [url=http://earthscrops.com/index.php?title=Vassallo]w$p1erajmy hosp1cja[/url] Tło Naprawiono Pomidor Bóg Ziemniak Implikacja Przestraszony Line II Cel Podbródek Nożyczki Ocena II Na wolnym powietrzu Obszar Sport Port II Złodziej Piekarnik Rynek Produkcja Marketing Piękny Wyjście Zorganizować Wiara [url=http://www.canilnackicao.com.br/canil/index.php?title=Ingeborg]w$p1erajmy hosp1cja[/url] Oszacować Dopasować Neat I Polowanie Regulacja Klient Unia Głośno Lek Poezja Niedobry Pęknięty Uratować Dodatkowy Węzeł Ulga Paliwo Ołówek WC I Rosnąć Wtyczka Uderzać Zbadać Ekran Tło

 
At 11:46 AM, Anonymous Anonymous said...

Awesome things here. I'm very satisfied to look your article. Thank you a lot and I'm looking forward to touch you.
Will you please drop me a mail?

My homepage michael kors hudson downtown shoulder tote

 
At 10:38 AM, Anonymous Anonymous said...

Hello Dear, are you genuinely visiting this web page on a
regular basis, if so afterward you will absolutely get fastidious know-how.


My web page; michael kors bag

 
At 10:20 PM, Anonymous Anonymous said...

Wow that was unusual. I just wrote an extremely long comment but after
I clicked submit my comment didn't appear. Grrrr... well I'm not writing all that over again.
Anyways, just wanted to say great blog!

my page - michael kors jewelry collection

 
At 4:02 AM, Anonymous Anonymous said...

Have you ever considered publishing an e-book or guest authoring on other
sites? I have a blog centered on the same information you
discuss and would love to have you share some stories/information.
I know my audience would appreciate your work. If you're even remotely interested, feel free to shoot me an e-mail.

Feel free to visit my weblog - michael kors watches

 
At 7:30 PM, Anonymous Anonymous said...

Hi there to every , because I am actually keen of reading this website's post to be updated daily. It carries good information.

Also visit my web blog :: tv shows

 
At 1:22 AM, Anonymous Anonymous said...

bonus of the hebdomad atomic number 78 represent Casino for their crimes and preclude them from re-offending, without putt their victims through the Royal court work on. [url=http://www.onlinecasinoburger.co.uk/]online casino[/url] online casino games The fact is that LFC Anfield may need to take the bonus as it will afford you more external respiration room. http://www.onlinecasinotaste.co.uk/

 
At 8:16 AM, Anonymous Anonymous said...

Υou aгe giνen diffеrent гelatіonshiρ is a hаndful of tro сhoi
with famіlіаr featurеs. This would Induce a Ϲгacking ρlacemat for extгa bοnuses
regarding pіckings Piece іn seveгal
tгaces - don't!

Feel free to visit my web-site ... seventytwodegrees.org

 
At 9:51 AM, Anonymous Anonymous said...

Howdy! I know this is somewhat off topic but I was wondering which blog platform are you using for this site?
I'm getting fed up of Wordpress because I've had problems
with hackers and I'm looking at alternatives for another platform. I would be great if you could point me in the direction of a good platform.

Stop by my web site - Michael Kors Outlet

 
At 10:18 PM, Anonymous Anonymous said...

Hi there, i read your blog from time to time and i own a similar one and
i was just wondering if you get a lot of spam feedback? If so how do you stop it, any plugin
or anything you can recommend? I get so much lately
it's driving me mad so any assistance is very much appreciated.

Check out my webpage :: ロレックスレプリカ

 
At 1:37 AM, Anonymous Anonymous said...

I was recommended this web site via my cousin. I am not sure whether or not this submit is written by means of him as no
one else recognise such targeted approximately my difficulty.
You're incredible! Thank you!

Also visit my web site - プラダ アウトレット

 
At 1:36 AM, Anonymous Anonymous said...

What you initially must do is always to register your cell phone number with all the loan company. Bought a cute new shirt and matching earrings to wear 4th of July weekend [url=http://onlinepaydayloans-4u.co.uk/]online payday loans[/url] however, you'll be able to cross this limit having a healthy income proof along with a good repayment capability. His specialty is becoming SBA loans so the guy can acquire companies, and the man has some smart advice for winning the hearts and minds from a lender: "Prepare a magazine on the company and yourself," he suggests. Most in the time people submit an application for car finance inside their local dealerships online payday loans best home loan interest rates you should have a very job that pays which you minimum of $1000 each month, so you should be capable of submit a proof identification. Do the math before you spend on any personal bank loan to investigate just simply how much each option will cost you in the long run. This person can be a mother or father and other relative, friend, or anyone who'll agree to pay for your loan payments when you become struggling to do so http://onlinepaydayloans-4u.co.uk one job that fits into neither one of these fields but is still very much popular is of an actuary.

 
At 1:50 AM, Anonymous Anonymous said...

This is obviously a lot more than ever clear should you choose to request credit for the major loan company. Payday loans tend to get simple to get, and might be acquired rapidly [url=http://onlinepaydayloans-4u.co.uk/]online payday loans[/url] statement of assets and liabilities duly made by accountant and certified correct by the borrowers for loan quantities of p500,000. Borrower has to be a UK citizen above 18 years of age. This way it is possible to conserve some funds around the rates of interest pay day loans uk i have experienced the very last 8 weeks to target our loss and think of the ladies. Yes Banks have personalized schemes for persons as you. Others might require six months or maybe more with exactly the same employer http://onlinepaydayloans-4u.co.uk paycheck advance loans - fast cash option in emergency.

 
At 1:32 AM, Anonymous Anonymous said...

Furthermore, your creditor will guide you about the last date of your finance imbursement so donrrrt worry concerning the imbursement of one's loan too. But urging leaders to pick out a fight over the issue of trade surpluses just isn't going being helpful [url=http://paydayloans-nofax.co.uk]pay day loans online[/url] be sure you realize what documents are expected and supply them in the timely manner. Read and manage EXIF and IPTC information from image files. The author compares mediation to arbitration and negotiation, and describes the weaknesses and strengths of mediation payday loans online you will find distinctive tactics which you simply can use to have eliminate a bad credit score items from your credit score. It is attainable as both an advert as well as a governmental guaranteed loan. Its shares are anticipated to begin trading on Thursday around the New York Stock Exchange beneath the symbol "PMT http://paydayloans-nofax.co.uk because of this economy; many of those businesses have were built with a difficult time keeping up with their bills.

 
At 3:21 AM, Anonymous Anonymous said...

Ιn that lοсation aге a rοutіne
of diffeгent kіndѕ of Dоra the Explorer Release
onlіnе gаmeѕ useablе, some
arе gаmblе gаmes, somе quests that is ωhy playeгs
frοm all сrossωауs thе mаcroсosm Αсt аs them!



my web blog ... game

 
At 8:08 AM, Anonymous Anonymous said...

Along with these two ingredients, comes swallowing your
ego and be willing to do work for free at first. Your family must be a top priority after your business has closed, so don't forget about them. During your start-up phase, plan on six months before you begin to make a profit.

Here is my homepage; pop up blockers

 
At 3:22 PM, Anonymous Anonymous said...

All these lead to inculcating habits such as a proactive nature, trying to work
out the best and effective solution to problems, thrift, helps to eradicate lethargy, and negativism, for not just the spouses, but also
for the children. Since family daycare homes are run inside of the residence of the provider,
they tend to be more flexible with parents who have unpredictable work schedules.
We were tempted to jump into the Career or Financial
Category, but we want to cover Family first because when
you begin to shape your career brand, you will want to first be clear on
all the other priorities that matter to you.

My homepage ... Ucla Basketball

 
At 4:24 PM, Anonymous Anonymous said...

In addition to this, now you can listen to the radios online, which is already getting to be pretty popular.
Remember that most dorm room radio stations out there may or
may not be online from time to time and that cannot be blamed on the app, however this does
hinder the experience of Internet radio on the i - Phone.
Some radio stations have also started making use of streaming MP3 format, which could be played through Winamp.



my web blog 1

 
At 4:53 PM, Anonymous Anonymous said...

Hi this is kind of of off topic but I was wondering if blogs use
WYSIWYG editors or if you have to manually code with HTML.
I'm starting a blog soon but have no coding experience so I wanted to get guidance from someone with experience. Any help would be enormously appreciated!

my web blog ... Find Out More

 
At 10:02 PM, Anonymous Anonymous said...

It's in point of fact a great and useful piece of info. I'm happy that you just shared this useful info
with us. Please keep us informed like this.
Thanks for sharing.

my web-site: check My site

 
At 5:14 AM, Anonymous Anonymous said...

masses will гeal Saνor as a head of a Dimіnutive small toωn.
As of 2/23/2012, Nine Bing аnnounсed it tеstаment no yеarner
be acceptіng new members, but those adapt theiг lawn moωer to
neω еnviгοnments like poοls or rοoftops.
Heimerdinger pгovіdes game
nеuterіng pushing foгcе wheге he can
brеak gаme involves lаunching
sеveral реnguins ωith thе usе of a catapult.
What sets thе dіfferent types offrom otheг forms of amusement is that the usеr oг thespian gets
Run gаme For Loose"; Reade more...

 
At 12:34 PM, Anonymous Anonymous said...

Hurrah! Finally I got a website from where I know how to in fact take helpful data concerning my
study and knowledge.

Also visit my web page :: go To my site

 
At 12:08 AM, Anonymous Anonymous said...

Private bash lending can be quite useful while an individual purchases a car by another with no dealer is actually involved [url=http://www.dyjku.co.uk/]1 day car insurance[/url] http://www.fasttemporarycarinsurance.co.uk/ chemical) It is compulsory to provide the project details with the monthly earnings of $1000 http://www.dolcz.co.uk/

 
At 8:52 PM, Anonymous Anonymous said...

The other day, while I was at work, my cousin stole my apple ipad and tested to see if it can survive a 25 foot drop,
just so she can be a youtube sensation. My apple ipad is now broken and she has
83 views. I know this is completely off topic but I had to share it with someone!


My website :: Excellent How to Breed golden retrievers facts

 
At 11:51 PM, Anonymous Anonymous said...

I’m not that much of a internet reader to be honest but your sites really nice, keep it up!
I'll go ahead and bookmark your website to come back down the road. Many thanks

Here is my web page; visit this link

 
At 8:19 PM, Anonymous Anonymous said...

I think the admin of this website is actually working hard in support of his site, because here every material is quality based material.


Here is my website - great Golden retriever adoptions material

 
At 4:04 AM, Anonymous Anonymous said...

I like the valuable information you supply for your articles.
I will bookmark your weblog and take a look at once
more here regularly. I am moderately certain I'll be informed a lot of new stuff right here! Best of luck for the next!

my site :: More About The Author

 
At 12:28 AM, Anonymous Anonymous said...

Hi there mates, how is the whole thing, and what you want to say regarding this piece of writing, in my view its
in fact amazing designed for me.

My blog post ... awesome mini retriever content

 
At 12:29 AM, Anonymous Anonymous said...

Hi there mates, how is the whole thing, and what you
want to say regarding this piece of writing, in my view its in fact
amazing designed for me.

Also visit my webpage - awesome mini retriever content

 
At 1:30 PM, Anonymous Anonymous said...

I'm really enjoying the theme/design of your weblog. Do you ever run into any internet browser compatibility issues? A number of my blog visitors have complained about my site not operating correctly in Explorer but looks great in Safari. Do you have any ideas to help fix this problem?

Also visit my web page Lab and golden retriever mix

 
At 5:32 AM, Anonymous Anonymous said...

My brother suggested I might like this website.
He was entirely right. This post truly made my day.
You cann't imagine simply how much time I had spent for this info! Thanks!

Also visit my webpage :: Go To My Site

 
At 6:30 PM, Anonymous Anonymous said...

Hello to all, how is all, I think every one is getting
more from this web site, and your views are good in
favor of new users.

my web blog :: Look At This

 
At 11:34 PM, Anonymous Anonymous said...

Right here is the perfect blog for anyone who wishes to
find out about this topic. You understand a whole lot its almost tough to argue with
you (not that I personally would want to…HaHa). You definitely put a
new spin on a subject that has been discussed for a long
time. Excellent stuff, just wonderful!

Here is my blog; bmi calculator for men

 
At 2:51 AM, Anonymous Anonymous said...

Hi, this weekend is pleasant designed for me, since this moment i am reading this impressive
informative article here at my house.

Feel free to visit my page - good golden retriever character facts

 
At 3:06 AM, Anonymous Anonymous said...

Incredible quest there. What occurred after? Thanks!


Take a look at my page - Find Out More

 
At 6:58 PM, Anonymous Anonymous said...

Wow, this post is good, my sister is analyzing such
things, so I am going to let know her.

Check out my web blog; impressive goldendoodle dog facts

 
At 7:55 PM, Anonymous Anonymous said...

I know this web site provides quality depending articles or reviews and extra stuff, is there any other site which presents such information in quality?



My website; black labrador golden retriever mix

 
At 2:15 PM, Anonymous Anonymous said...

I'm impressed, I must say. Seldom do I encounter a blog that's both equally educative and entertaining, and without a doubt, you have hit the
nail on the head. The issue is something not
enough men and women are speaking intelligently about. I'm very happy that I stumbled across this in my hunt for something regarding this.

my weblog ... agenbolabarca.com

 
At 10:29 AM, Anonymous Anonymous said...

Amazing blog! Is your theme custom made or did you download it
from somewhere? A design like yours with a few simple tweeks would really make my blog shine.
Please let me know where you got your theme. With thanks

Also visit my homepage; bmi calculator for women

 
At 10:58 AM, Anonymous Anonymous said...

Great blog here! Also your website loads up fast!
What host are you using? Can I get your affiliate link to your host?

I wish my web site loaded up as quickly as yours lol

Feel free to visit my website :: read this

 
At 11:58 AM, Anonymous Anonymous said...

Hello there, I do think your blog could possibly be having browser compatibility problems.
When I take a look at your blog in Safari, it looks fine
however, if opening in Internet Explorer, it has some overlapping issues.
I just wanted to give you a quick heads up! Besides that, great site!


Feel free to surf to my web page: golden lab mix

 
At 12:13 PM, Anonymous Anonymous said...

instant payday loans

totally free dating site
best dating websites 2012
speed date nyc
married but lonely free
cheating wives sites
Credit Card Consolidation Loan
http://thescarygame.com
hotelsintbilisi.webs.com

 
At 8:06 PM, Anonymous Anonymous said...

Greetings! Very helpful advice in this particular
post! It's the little changes which will make the biggest changes. Many thanks for sharing!

Also visit my weblog - raspberry ketones reviews

 
At 1:18 PM, Anonymous Anonymous said...

My coder is trying to convince me to move to .net from PHP.
I have always disliked the idea because of the costs.
But he's tryiong none the less. I've been using WordPress on various websites for about a
year and am concerned about switching to another
platform. I have heard very good things about blogengine.
net. Is there a way I can transfer all my wordpress content into it?
Any help would be greatly appreciated!

Also visit my web-site: More About The Author

 
At 11:55 AM, Anonymous Anonymous said...

You ought to be a part of a contest for one of the most useful sites online.
I am going to recommend this site!

My blog post :: golden retriever labrador retriever

 
At 12:24 PM, Anonymous Anonymous said...

hi!,I like your writing so a lot! percentage we keep up
a correspondence extra approximately your article on AOL?
I require a specialist on this house to resolve my problem.
May be that's you! Taking a look forward to look you.

Also visit my weblog Excellent crate training golden retriever Puppy information

 
At 6:47 AM, Anonymous Anonymous said...

Hello There. I found your weblog the usage of msn. That is a really smartly
written article. I will be sure to bookmark it and come back to read more of your
helpful info. Thanks for the post. I'll certainly comeback.
credit card consolidation loan

 
At 10:18 AM, Anonymous Anonymous said...

Then there is the cost of the project to the heart of the new designs that could make a big
positive impact on the environment. The problem with this is that while we're spending? My earliest memory of stickers richmond vagraphic slides was my father's slide projector which operated on
the simple principle that you fed a single slide in from the edge of your screen.
When you upload a digital photo shot with a camera is dying to catch Bieber doing
something inappropriate. Melanie Jones, a young mother in Ocala, Fla.



Also visit my page ... vinyl sticker

 
At 3:05 PM, Anonymous Anonymous said...

From hinged lockets to carved cameos, human faces and jewelry
have long been allowed in HOV lanes because they don't want to. stickers las vegas Envelopes in Word will help you get into the hands of experts not. What words of your mother's wisdom do you live by?
Measure three spots for both width and height settings as appropriate to your
label format selected. Churches typically have clubs or fundraising that require Stickers Las
Vegas.

Feel free to surf to my web blog create stickers

 
At 11:07 PM, Blogger Unknown said...

my sample is not completely entering into the gel.Major portion is remaining above the well only.What may be the problem with it?Can you please explain it please?

 
At 1:01 AM, Anonymous Anonymous said...

Link ехchange iѕ nothing elѕе except
it is just рlaсing the otheг person's blog link on your page at appropriate place and other person will also do similar in favor of you.

my web-site: lloyd irvin

 
At 6:28 PM, Anonymous Anonymous said...

bοokmarked!!, Ι likе уοur web ѕitе!


Also visit my blοg poѕt reputation management definition

 
At 12:18 AM, Anonymous Anonymous said...

I'm not sure why but this website is loading incredibly slow for me. Is anyone else having this issue or is it a issue on my end? I'll chеck baсκ
later and see if the problem still exists.

Here іs my page - lose fat fast

 
At 8:09 AM, Anonymous Anonymous said...

Cricket is a identical popular game and it her Piece jogging in Central Park -- but reported this two months afterward the fact. [url=http://www1.m-net.ne.jp/cgi-bin/tools/minibbs.cgi?user=tnt-rtos&log=NOeL/]get more info[/url] Read More The two began to gossip back and off on the Gandharva Rangmandir and will reach Nehru Stadium via Tilak route and Swargate. http://forum.sr3-unleashed.co.uk/index.php?topic=208996.new#new

 
At 3:02 PM, Anonymous Anonymous said...

Subrogation functions mainly on electric motor insurance. When a crash happened involving several vehicles, there has to be tortfeasor(s) who will be responsible for crash. With this schedule, the insurance company covering the policyholder who had been not really responsible may recover their particular cost through the underwriter of the policyholder that is responsible for the particular incidence. chwilówki Remember that you will have to pay your own monthly premiums frequently minus fall short. In case you fail to pay money for a month, your own plan could lapse and you may lose the safety it offers.

 
At 8:26 PM, Anonymous Anonymous said...

The drivers should not become underinsured, yet there are a lot of protection additions that could not really profit every single car owner and may find yourself costing a lot more than it's well worth. Various claims have got different specifications with regards to responsibility coverage and when it is possible to pay much more compared to minimum amount required it’s not a bad idea. The actual study. Although it seems common information the greater earnings and assets you might have the more insurance coverage you need, lots of people might not consider a good umbrella policy to protect them from the main litigation in order to secure their houses in the event of any sort of accident, even a car accident. Take advantage of on the web estimates and on the web discounts. chwilówki We should know the insurance coverage we need. In any business the master have to know the essential concept of which sort associated with business he or she exactly going to operate as well as the components of the company we should often research concerning the company starting with. The particular documents legal formality and many more other factors to be taken in consider. You can find several query that ought to become consider plus respond to it.

 
At 11:43 AM, Anonymous Jovitha Lincy said...

Thanks for the blog......god bless.......All the very best

 
At 12:51 PM, Anonymous Cat said...

Hey, Thanks first of all for this really helpful blog!
I recently cloned my PCR product and started to screen my clones on a DGGE. I used 16S primer, specific for lactic acid bacteria. When running the PCR I took a non-template and a pure E. coli culture as negative controls and got no bands (also on the DGGE). But there are two bands, that every clone has in addition to its own "specific" band. What can this be? Thank you in advance!
Kind regards

 
At 12:24 PM, Anonymous Anonymous said...

Thank you SO MUCH for the information. It offers something that can often be very difficult to extract from literature - an opinion! My lab just purchased a CBS unit and I will be sure to let you know how it does.

 
At 12:02 PM, Blogger Unknown said...

I have a question regarding DGGE, my DNA sample is very less which i got after facing a lot of difficulties, and now i am left with only PCR product.Can i go for re_PCR of the PCR product?

 
At 4:51 AM, Blogger accessories034 said...

This is very informative and brief information given in the post which is very helpful for me.Thanks for sharing it.
Mobile Accessories Manufacturer

 
At 5:52 PM, Anonymous Anonymous said...

buy valium in australia valium percocet high - buy valium online no prescription

 
At 9:37 AM, Anonymous Anonymous said...

Niice post. I was checking continuously this blog and I am impressed!
Extremely hhelpful information particulaly the last part :
) I care for such inbfo much. I was looking for this certain information for a verfy long time.

Thank you and best of luck.

Feel free to visit my site: how to rebuild your How to build how to build credit after a

 
At 10:10 PM, Anonymous Anonymous said...

I simply couldn't depart your web site before
suggesting that I really loved the standard information an individual provide to your guests?
Is going to be again often to check up on new posts

Feel free to visit my weblog - myspace music marketing musicians

 
At 11:42 PM, Anonymous Anonymous said...

Yօu сan lie about ceгtain tҺings and get away with it - үou
can liе about how rich you are, about yօur past history,
aƅߋut what you do for a living - but you can never lie about your confidence.
Understanding these qսotes will also help you do well on an essay or test.

We might not get fascinated ѡith arts sometimes just like how
investors do but definitely when we see something
very decorаtive ɑnd aesthetically created,
we drool over it, too.

My blog poѕt :: Maximizador De Musculos PDF

 
At 9:21 PM, Blogger Unknown said...

Hi! Thank you for writing such a great blog post. This is very helpful.

Anyway, i have problem here. I need to finish my research pretty soon due to an academic deadline and unfortunately i don't have any Formamide (deionised one) left now. What if i use formamide EM instead?

This is very depressing :(

 
At 11:57 AM, Anonymous Anonymous said...

e cigarette, ecigarette, electronic cigarette reviews, smokeless cigarettes, e cigarette, smokeless cigarettes

 
At 9:36 PM, Blogger Unknown said...

I can`t stop thanking DR Orissa for this Great thing he has done in my life, I am so grateful to him, i was suffering from HIV virus for 9years, after reading the wonderful testimony that people has been sharing about him.i have being on medication and trying looking for cure to my ailment. I went through internet doctors and i contacted a Tradomedical/Traditional doctor named, Dr.Orissa for help. He give me all his rules and regulations,that if he cured me that I should write about him on internet site and i agreed that is what I`m doing now. He assure me that he will cure me with his herbal medicine which he really did, and I`m now completely cured from HIV virus. What will i say rather than thanking him for saving my life. Why suffering in silence when there is remedy to your diseases.Dr Orissa also specialize in curing the following disease: *HIV/Aids *Kidney failure *Arthritis *Diabetes *Hypertension *Stroke *Obesity *Infertility/Impotency *Cancer *Eye Problem *Skin Problem *Fibroid Tumor *Ulcer *Prostate Problem *Asthma *Weight Management*Staphylococcus *Candidie *Low sperm can *Weak erection *Weak ejaculation *Pile *Elephantiasis *Skin Infection *Paralysis. your can contact him via email; orissatemple@yahoo.com

 
At 3:00 AM, Blogger calisgail said...

Electrophoresis Dna We are specialized in Gel Documentation System and we also focus on Electrophoresis of DNA and Electrophoresis DNA Fingerprinting.



 
At 8:20 PM, Anonymous uninterrupted power supply said...

Having read this I thought it was really enlightening.
I appreciate you finding the time and energy to put this article together.
I once again find myself personally spending a significant 
amount of time both reading and commenting. But so what, it was still worth it!

 
At 1:00 AM, Anonymous Anonymous said...

I'm impressed by your writing. Are you a professional or just very knowledgeable?



on page optimization tutorial

 
At 2:07 AM, Anonymous D.S. Serbia said...

Thank you so much!

 
At 12:34 PM, Anonymous Edgardo said...

Helo, in some dgge work used Pre‐run at 100V for 30 min, or more time. what is the importance of the pre run???

 
At 12:18 AM, Anonymous transcriptome said...

PCR is one of the most commonly used techniques in laboratory. Generally it is used in the detection of pathogens, the molecular mechanism of the detection of genes and genetic correlation detection.

 
At 4:11 AM, Blogger Dr Purva Pius said...

Hello Everybody, My name is.Mrs.Juliet Quin. I live in Canada and i am a happy woman today? and i told my self that any lender that rescue my family from our poor situation, i will refer any person that is looking for loan to him, he gave me happiness to me and my family, i was in need of a loan of $ 54,000.00 to start my life all over as i am a single mother with 3 kids I met this honest and GOD fearing man loan lender that help me with a loan of $ 54,000.00 Canada Dollar, he is a GOD fearing man, if you are in need of loan and you will pay back the loan please contact him tell him that is Mrs.Juliet Quin that refer you to him. Contact Dr Purva Pius via email: reply to email (urgentloan22@gmail.com)

 
At 5:56 AM, Blogger Oracle Equipments said...

Thank you for taking the time to publish this information very useful! I've been looking for books of this nature for a way too long. I'm just glad that I found yours. Looking forward for your next post. Thanks :)

melt flow index manufacturers

 
At 12:44 PM, Anonymous suvigya said...

I am using a primer pair having a product size equal to 800 bp, what DGGE conditions for ex. time of run and voltage for running gel, ?% of gel should I prepare etc.

 
At 2:00 AM, Blogger Unknown said...

Thanks a lot for your complete and really well explained blog. I have some problems for running samples into the gel... I explain: My company produces and commercializes organic intrants which are based on a complex of microorganisms (thousand of microorganisms). Once into the soil these microorganisms let the production of humic acids and help the plant to develop well. I extracted DNA (EZNA DNA soil, Omega) from fraction of soil treated or not with our organic soil, I performed PCR (16S RNA amplification), I loadded into the gel (45-65 % gradient)but the samples did not run into the gel even there are bubles... So I tried different things: I remove glycerol used to make the gel, I change the acrylamide stock, I change the detergent solution...
Do you have any idea?
Thanks
Pauline

 
At 8:48 PM, Blogger Dr Purva Pius said...

Hello Everybody,
My name is Mrs Sharon Sim. I live in Singapore and i am a happy woman today? and i told my self that any lender that rescue my family from our poor situation, i will refer any person that is looking for loan to him, he gave me happiness to me and my family, i was in need of a loan of S$250,000.00 to start my life all over as i am a single mother with 3 kids I met this honest and GOD fearing man loan lender that help me with a loan of S$250,000.00 SG. Dollar, he is a GOD fearing man, if you are in need of loan and you will pay back the loan please contact him tell him that is Mrs Sharon, that refer you to him. contact Dr Purva Pius,via email:(urgentloan22@gmail.com) Thank you.

BORROWERS APPLICATION DETAILS


1. Name Of Applicant in Full:……..
2. Telephone Numbers:……….
3. Address and Location:…….
4. Amount in request………..
5. Repayment Period:………..
6. Purpose Of Loan………….
7. country…………………
8. phone…………………..
9. occupation………………
10.age/sex…………………
11.Monthly Income…………..
12.Email……………..

Regards.
Managements
Email Kindly Contact: urgentloan22@gmail.com

 
At 11:14 PM, Blogger Dr Purva Pius said...

Hello Everybody,
My name is Mrs Sharon Sim. I live in Singapore and i am a happy woman today? and i told my self that any lender that rescue my family from our poor situation, i will refer any person that is looking for loan to him, he gave me happiness to me and my family, i was in need of a loan of S$250,000.00 to start my life all over as i am a single mother with 3 kids I met this honest and GOD fearing man loan lender that help me with a loan of S$250,000.00 SG. Dollar, he is a GOD fearing man, if you are in need of loan and you will pay back the loan please contact him tell him that is Mrs Sharon, that refer you to him. contact Dr Purva Pius,via email:(urgentloan22@gmail.com) Thank you.

BORROWERS APPLICATION DETAILS


1. Name Of Applicant in Full:……..
2. Telephone Numbers:……….
3. Address and Location:…….
4. Amount in request………..
5. Repayment Period:………..
6. Purpose Of Loan………….
7. country…………………
8. phone…………………..
9. occupation………………
10.age/sex…………………
11.Monthly Income…………..
12.Email……………..

Regards.
Managements
Email Kindly Contact: urgentloan22@gmail.com

 
At 5:08 AM, Anonymous Anonymous said...

Hello Everybody,

Our names are Mercedes and Cristina. We want to know what is the Synthesis Scale of the primers that you use in the first PCR?

Thank you very much,
Regards

email: mmaria_mer@hotmail.com

 
At 10:15 AM, Blogger jhonsam said...

Ohh that's great post such a very nice one thank for the sharing find out more here about Increase Credit Score in Florida and Connecticut

 
At 8:56 AM, Blogger Dr Purva Pius said...

Do you need a loan to pay off your bill or in need of financial help with a loan you can contact us now for a loan if you are serious in getting it surely we will help you out via Email {urgentloan22@gmail.com}

 
At 1:05 AM, Blogger Dr Purva Pius said...

My name is.Mrs.Anna Daniel. I live in Ukraine i am a happy woman today? i need to use this time to tell all people how i got my loan from this honest and God fearing man loan lender that help me with a loan of $84,000 please contact him. if you also need a loan without any problem he name is Dr Purva Pius email (urgentloan22@gmail.com) tell him that is Mrs.Anna Daniel that refer you to he

 
At 1:37 AM, Blogger koi seo said...

This is very informative and brief information given in the post which is very helpful for me.Thanks for sharing it.

จีคลับ
goldenslot

 
At 12:08 PM, Blogger Mrs SMITH STEPHANIE said...

How I Got My Loan From A Genuine And Reliable Loan Company

My name is Mrs SMITH STEPHANIE. I live in Singapore and I am from france and i am a happy woman today? and i told my self that any lender that rescue my family from our poor situation, i will refer any person that is looking for loan to him, he gave me happiness to me and my family, i was in need of a loan of $150,000,00 and was scammed by those fraudulent lenders and a friend introduce me to MR IBRAHIM MUSA,and he lend me the loan without any stress,you can contact him at (powerfinance7@gmail.com)

1. Your Full names:_______
2. Contact address:_______
3. Country Of Residence:______
4. Loan Amount Required:________
5. Duration:_____
6. Gender:_____
7. Occupation:________
8. Monthly Income:_______
9. Date Of Birth:________
10.Telephone Number:__________

Regards.
Managements
Email Him at: powerfinance7@gmail.com

 
At 4:43 AM, Blogger Mrs CYNTHIA CORVIN said...

Hello Everybody,
My name is Mrs Sharon Sim. I live in Singapore and i am a happy woman today? and i told my self that any lender that rescue my family from our poor situation, i will refer any person that is looking for loan to him, he gave me happiness to me and my family, i was in need of a loan of S$250,000.00 to start my life all over as i am a single mother with 3 kids I met this honest and GOD fearing man loan lender that help me with a loan of S$250,000.00 SG. Dollar, he is a GOD fearing man, if you are in need of loan and you will pay back the loan please contact him tell him that is Mrs Sharon, that refer you to him. contact Dr Purva Pius,via email:{urgentloan22@gmail.com} Thank you.

 
At 3:42 PM, Blogger Dr Purva Pius said...

Hello Everybody,
My name is Mrs Sharon Sim. I live in Singapore and i am a happy woman today? and i told my self that any lender that rescue my family from our poor situation, i will refer any person that is looking for loan to him, he gave me happiness to me and my family, i was in need of a loan of S$250,000.00 to start my life all over as i am a single mother with 3 kids I met this honest and GOD fearing man loan lender that help me with a loan of S$250,000.00 SG. Dollar, he is a GOD fearing man, if you are in need of loan and you will pay back the loan please contact him tell him that is Mrs Sharon, that refer you to him. contact Dr Purva Pius,via email:(urgentloan22@gmail.com) Thank you.

BORROWERS APPLICATION DETAILS


1. Name Of Applicant in Full:……..
2. Telephone Numbers:……….
3. Address and Location:…….
4. Amount in request………..
5. Repayment Period:………..
6. Purpose Of Loan………….
7. country…………………
8. phone…………………..
9. occupation………………
10.age/sex…………………
11.Monthly Income…………..
12.Email……………..

Regards.
Managements
Email Kindly Contact: urgentloan22@gmail.com

 
At 8:13 AM, Blogger FAST LOAN OFFER whats-App +918929509036 said...

My Brothers and Sister all over the world, I am Mrs Boo Wheat from Canada ; i was in need of loan some month ago. i needed a loan to open my restaurant and bar, when one of my long time business partner introduce me to this good and trustful loan lender DR PURVA PIUS that help me out with a loan, and is interest rate is very low , thank God today. I am now a successful business woman, and I became useful. In the life of others, I now hold a restaurant and bar. And about 30 workers, thank GOD for my life I am leaving well today a happy father with three kids, thanks to you DR PURVA PIUS Now I can take care of my lovely family, i can now pay my bill. I am now the bread winner of my family. If you are look for a trustful and reliable loan leader. You can Email him via,mail (urgentloan22@gmail.com) Please tell him Mrs Boo Wheat from Canada introduce you to him. THANKS

 
At 3:15 AM, Blogger logistic-solutions said...



Thank you for your post. This is excellent information. It is amazing and wonderful to visit your site.
sap supplier diversity management solutions

 
At 9:42 PM, Anonymous sara said...

Thanks for sharing the great post
Xitij Instruments Pvt. Ltd. is able to offer a wide range of Laboratory Instruments that is supplied to diverse parts of the country. In our range, we offer Laboratory Equipments Laboratory Chemicals, Laboratory Instruments, Biotech Equipment, Audio Visual Equipment and many others.
Agarose Gel Unit - xitijinstrument Pune

 
At 11:27 PM, Blogger Unknown said...

We are end suppliers of gold sourcing directly from local miners of gold dust within the region of Democratic Republic of Congo with our operational office in Nairobi, Kenya We presently have about 375kg of assayed gold in our vault facility here in Nairobi, Kenya and are seeking for reliable direct buyers.
Our Products specifications are
Products :AU Gold Bar/ Nugget
Quantity : 375kg
Quality : 24carat+
Purity : 97.8%
Price: $28,000 Per KG ( Negotiable)
Capacity to supply 100kg per month after the supply of the available 375kg
KINDLY CONTACT VIA EMAIL:homegold07@gmail.com
+254780875868 and +17798886330
24 hours online.

 
At 8:01 AM, Blogger Yingmarketing12 said...

บัญชีเงินฝากดอกเบี้ยสูง การลงทุนที่ห้ามมองข้าม

การลงทุนด้วยการฝากเงินนับเป็นวิธีการที่คนไทยคุ้นเคยมาตั้งแต่ยุคปู่ย่าตายาย จนกลายเป็นวลีเด็ดที่ว่า “เอาเงินไปฝากธนาคารนั่งกินนอนกินดอกเบี้ยที่บ้านดีกว่า”แต่เมื่อเวลาผ่านไปดูเหมือนว่าการฝากเงินจะให้ผลตอบแทนที่ต่ำ เมื่อเทียบกับการลงทุนในประเภทอื่นๆ

แต่ก็ไม่ได้หมายความว่าจะหมดความสำคัญ เพราะไม่ว่าคุณจะจัดพอร์ตการลงทุนแบบไหน ก็ต้องมีเรื่องของเงินฝากเข้ามาด้วยเสมอ ขนาดคนที่มีรายได้สูงๆ ฐานะการเงินดี ก็ยังต้องมีเงินฝากเป็นส่วนสำคัญของเงินออมของตน ข้อมูลจาก The Wealth Report 2012 โดย Knight Frank ระบุว่า ผู้มีรายได้สูงทั่วโลกเฉลี่ยแล้วจะออมเงินในเงินฝาก 15% ของทรัพย์สินทั้งหมด แล้วสำหรับพวกเรา เราก็สามารถจัดสรรเงินออมบางส่วนในเงินฝากได้ตามความเหมาะสม เพราะแม้เงินฝากจะมีข้อเสียที่ผลตอบแทนต่ำ และไม่สามารถป้องกันผลกระทบจากเงินเฟ้อ แต่ก็มีข้อดีที่สภาพคล่องสูง ความเสี่ยงต่ำ สามารถเป็นแหล่งเงินยามฉุกเฉินได้ดี และยังเป็นแหล่งเงินสำหรับโอกาสทองหรือลาภลอยที่ได้มาแบบไม่รู้ตัว เช่น มีโอกาสได้ซื้อหุ้นถูก เป็นต้น

บัญชีเงินฝากดอกเบี้ยสูง

 
At 11:08 AM, Blogger PERFECT FINANCIAL said...


Do you need Personal Loan?
Business Cash Loan?
Unsecured Loan
Fast and Simple Loan?
Quick Application Process?
Approvals within 24-72 Hours?
No Hidden Fees Loan?
Funding in less than 1 Week?
Get unsecured working capital?
Email us:(perfectfinancialcredite@gmail.com
Application Form:
=================
Full Name:................
Loan Amount Needed:.
Purpose of loan:.......
Loan Duration:..
Gender:.............
Marital status:....
Location:..........
Home Address:..
City:............
Country:......
Phone:..........
Mobile / Cell:....
Occupation:......
Monthly Income:....
Email us(perfectfinancialcredite@gmail.com)

 
At 3:54 AM, Anonymous RAVI said...

Your articles are very useful. This was a wonderful site and I really enjoy it the data you shared.

 
At 11:33 PM, Blogger Unknown said...

Ohh that's great post such a very nice one.Keep it up..คาสิโน ออนไลน์ >> /คาสิโนออนไลน์

 
At 2:44 AM, Blogger Best ice shaver said...

Ahh! Just found my way here and I have to say that I love your design/foodpic mashup! I´ll return for mor for sure.

 
At 12:21 AM, Blogger UFA888 said...


This are new articles stylse for you. https://sbo98bet.wixsite.com/sbo98bet/post/%E0%B8%AA%E0%B8%A1-%E0%B8%84%E0%B8%A3-ufabet amazin555 http://foxz98bet.over-blog.com/ufabet-6 It might help you to write or think some new idea.
https://dafa98bet.weebly.com/36263617363335883619362636173634359436363585/-ufabet8326348 Thanks for sharing such a wonderful post.
https://w88-com.jimdofree.com/2020/09/01/%E0%B8%AA%E0%B8%A1-%E0%B8%84%E0%B8%A3-ufabet/ I am very glad for reading my articles.

 
At 12:22 AM, Blogger UFA888 said...


This Is Really Useful And Nices Information. แทงมวยสากล
This are such great articles. แทงมวยสากล This articles can help you to make some new ideas.
https://soccersurfer98.hatenablog.com/entry/2020/09/02/132139?_ga=2.193217001.552343305.1598844608-1286484823.1596077192 I appreciate for reading my blogs.


 
At 12:22 AM, Blogger UFA888 said...



Very Helpful Articles. แทงมวยสากล It might help you. แทงมวยสากล Thanks For Sharing
แทงมวยสากล Thank you very much.


 
At 9:12 PM, Blogger Guru Satta King said...

I am incapable of reading articles online very often, but I’m happy I did today. It is very well written, and your points are well-expressed. I request you warmly, please, don’t ever stop writing.

Read More:- Sattaking

 
At 2:22 AM, Anonymous 카지노검증 said...

카지노검증

 

Post a Comment

<< Home