Product of Interest

Dear All -

I was at a VWR show last week and I saw a product that could be of interest to DGGE users who want to recover DNA from excised bands. The product is a "FlexTube", and as far as I can tell, you place the excised band in the tube, place the tube in a standard gel box, run for a while, and recover the DNA in the eluent. The product is sold by IBI Scientific (http://www.ibisci.com/), and for more information you can contact Patrick L. Mueller (plmueller@ibisci.com). The products at list price seem quite expensive, but perhaps viable for key samples.

Just a note: I am not affiliated with IBI scientific nor do I have any financial interest in the company. I also have not used this product. This posting is merely to alert DGGE users to a product of potential interest. I will be interested to know if users successfully employ this product.

Thanks, Stefan

New manuscript about reproducibility of GC clamps

One more thing to worry about!

A recent article of interest in FEMS Microbiology Letters:

Rettedal et al. 2010. GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles. DOI: 10.1111/j.1574-6968.2010.02097.x

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GC-clamp primer batches yield 16S rRNA gene amplicon pools with variable GC clamps, affecting denaturing gradient gel electrophoresis profiles
Elizabeth A. Rettedal, Sharon Clay, Volker S. Brözel.

Abstract
Fingerprinting methods such as denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene pools have become a popular tool for comparisons between microbial communities. The GC-clamp portion of primers for DGGE amplicon preparation provides a key component in resolving fragments of similar size but different sequence. We hypothesized that repeat syntheses of identical 40-base GC-clamp primers lead to different DGGE profiles. Three repeat syntheses of the same GC-clamp primer and two different GC-clamp primers directed at the V3–5 region of the 16S rRNA gene were compared. Genomic DNA of two separate soil bacterial communities and three bacterial species was amplified and resolved by DGGE. The DGGE profiles obtained with repeat-synthesized primers differed among each other as much as with alternate primers, for both soil DNA and pure single species. The GC-clamp portion of members of amplicon pools varied among each other, deviating from the design sequence, and was the likely cause for multiple bands derived from a single 16S rRNA gene sequence. We recommend procuring an oligonucleotide batch large enough to conduct an entire project. This should help to avoid any DGGE profile variations due to performance differences between repeat syntheses of GC-clamp oligonucleotide primers.

Interesting article comparing Ingeny to Biorad

-- IMMEDIATE LINK TO GUIDE --

-- LINK TO DGGE BOOK CHAPTER --

Hi Folks -
I just found this article in which the authors examine the effects of using different DGGE systems on the same samples.
Cheers,
Stefan


J. Ascher, M. T. Ceccherini, A. Chroňáková, J. Jirout, F. Borgogni, D. Elhottová, M. Šimek and G. Pietramellara.Evaluation of the denaturing gradient gel electrophoresis-apparatus as a parameter influencing soil microbial community fingerprinting. World Journal of Microbiology and Biotechnology. 10.1007/s11274-010-0349-z.


Abstract
We compared two denaturing gradient gel electrophoresis (DGGE) systems—DCode (Biorad, Hercules, CA, USA) and PhorU (Ingeny, Leiden, NL), performing community level 16S and 18S rRNA gene fragment-PCR-DGGE with total DNA extracted from upland pasture soil used for outdoor cattle husbandry. The methodological evaluation of the DGGE apparatus as parameter influencing DGGE fingerprinting, based on cluster analysis of soil bacterial and fungal community fingerprints, was made in terms of the resulting information about microbial community structures and their response to different degrees of cattle impact. Although the comparative DGGE analysis with different DGGE systems provided similar clustering of microbial community structures in correlation with the degree of cattle impact, our results suggest the DGGE system to be a factor influencing DGGE analysis. To our knowledge this is the first attempt to investigate the hypothetical impact of the DGGE system due to different technical characteristics, recommending the use of one and the same DGGE apparatus throughout an experiment, if the monitoring of microbial community structures requires multiple gel-to-gel analysis.

Link to Springer Version of Book Chapter

Please find a link to the Springer Version of the DGGE Book Chapter (For those of you who can access the full text!). If you don't have access, another version is available here.
Cheers,
Stefan

Developments in Anti-Smiling Technology

-- IMMEDIATE LINK TO GUIDE --

-- LINK TO DGGE BOOK CHAPTER --


Dear DGGE users -

There is a new product from Ingeny to reduce and/or prevent smiling for DGGE gels. For the Ingeny system, the product is both a new spacer and a special loading solution for the first and last lanes. Information regarding these items can be found here.
I spoke with Ingeny, and they are intending to make spacers for other DGGE systems.
The loading solution alone is not effective.

Looks like a nice breakthrough.

Best,
Stefan

DGGE Book Chapter

Dear Friends -

While it may seem that I haven't posted much lately, I have still been working on DGGE-related matters. Together with my colleagues Mary Beth Leigh and Josh Neufeld, I have written a book chapter entitled "Denaturing gradient gel electrophoresis (DGGE) for microbial community analysis". It has some background on the method, as well as detailed protocols for producing gels, primer sets for bacterial, archaeal and fungal community analysis, a list of all required reagents, comparison of three major DGGE makers, and troubleshooting. If this chapter is useful to you, please cite the following:

Green, S.J., Leigh, M.B. and Neufeld, J.D. 2009. Denaturing gradient gel electrophoresis (DGGE) for microbial community analysis. Pages 4137-4158 in: Timmis, K.N. (Ed)Handbook of Hydrocarbon and Lipid Microbiology. Springer (Heidelberg, Germany).

Currently, the chapter is available for download. There may be some slight differences between this and the final typeset chapter, but this should be marginal.

Cheers,
Stefan

SB buffer for DGGE?

I recently received this question:
Hi Stefan,
I am new to this blog and stumbled upon it. Have you used the SB buffer in the DGGE apparatus? If so what have your run times and concentrations been? I have been using SB buffer with standard agarose gels but I am still using TAE buffer in our DGGE set up at 150V for 10h. Just curious and interested in saving some time.
Dusty

-- My response:
No, I haven't tried it. I am now in a lab without a DGGE, so I haven't had a chance. Theoretically, it should work and it should significantly reduce the cost of making buffer. I doubt it will make a big difference in the running time, because the limitation on voltage in a DGGE doesn't have much to do with the gel heating up (as in agarose gel electrophoresis).

Some thoughts - make sure to use SB buffer when making the acrylamide stock solutions. If you (or anyone else) tries SB buffer in place of TAE, please email me about the results and I'll post it.

Cheers,
Stefan

P.s. I have received the following comment from Michael (see below). His response would indicate that SB buffer is NOT appropriate for DGGE. C'est la vie.

"In fact I also have quite bad experience with SB buffer. Apart from the worse separation, it seems that SB somehow modifies gel's structure, making it softer and more fragile. Moreover, gels submerged into the staining solution significantly increase their dimensions, which suggests some kind of inhibition of the poliacrylamide polimerisation."