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."

New NCBI BLAST function for primer design

I thought this would be of interest to the community. The BLAST site at NCBI now has a specific page for the design and testing of PCR primers (click here). It looks to be quite sophisticated if necessary (there is a pull down menu for more advanced options).

http://www.ncbi.nlm.nih.gov/tools/primer-blast/index.cgi?LINK_LOC=BlastHomeAd

DGGE standards

- IMMEDIATE LINK TO GUIDE -

A message at the Yahoo DGGE group indicates that a company in Japan (Nippon Gene Co., LTD.)is selling DGGE markers for a variety of different gradient conditions. I tracked down the website, and although most of the text is in Japanese, it is still fairly easy to figure out what products are what. Anyway, I'm including a link to the standards page. And, as always - I am not affiliated with this company in any way! Caveat emptor. And remember - you can always make your own standards if you put your mind to it.

Cheers,
Stefan

Cyanobacterial Article Available for Free, Online at ISME Journal

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Ok, my cyanobacterial article - "A salinity and sulfate manipulation of hypersaline microbial mats reveals stasis in the cyanobacterial community structure" is a featured (and therefore freely accessible) article in the current issue of the ISME Journal.
Enjoy.

Cheers,
Stefan

Post-doctoral position in Israel available

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Ok, this isn't strictly a DGGE issue, but you might get to use DGGE....!

If anyone is interested in pursuing a post-doctoral position in microbial ecology, I can highly recommend the following project, sponsored by several excellent scientists in Israel.

The project is a cooperation between the Israel's police Division of Identification and Forensic Science, the Minz Laboratory, the Gillor Laboratory and the Jurkevitch Laboratory.

The goal of the project is to develop a forensic tool for the analysis of soil samples using bacterial communities fingerprints. The post-doc fellow will be in charge of developing, applying and validating protocols for soil analysis using molecular markers, analyzing large numbers of samples, and building an adequate integrative approach for sample correlation. He/she will coordinate the work of students and technicians of the various groups. The research requires skills in molecular microbial ecology, bioinformatics and statistical analysis and biochemistry.

If interested, please contact: Prof. Edouard Jurkevitch (jurkevi@agri.huji.ac.il), Dr. Osnat Gillor (gilloro@bgu.ac.il) or Dr. Dror Minz (minz@agri.gov.il).

New Phylum Level Primer Sets for DGGE Analysis

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A manuscript (Muhling et al. 2008) entitled "Improved group-specific PCR primers for
denaturing gradient gel electrophoresis analysis of the genetic diversity of complex microbial communities" was just was published online-early at the ISME journal website. This looks to be a promising development for the field with primers sets targeting Alpha, Beta, and Gamma-proteobacteria, Firmicutes, Bacteroidetes, Planctomycetes, and Cyanobacteria.

Enjoy,
Stefan

New Publication in the ISME Journal

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While I don't intend to use the DGGE site for my own personal vehicle, some readers may be interested in my most recent publication. I have used cluster analysis of PCR-DGGE gels, band excision and sequencing, and colony-PCR screening of general bacterial clone libraries with cyanobacterial-specific primers to explore the effect of salinity and sulfate manipulations on cyanobacterial communities in hypersaline mats. The methodological approach may be of interest to many members of the DGGE community even if you are not working on cyanobacteria.

Green SJ, Blackford CA, Bucki P, Jahnke LL and Prufert-Bebout L. 2008. A salinity and sulfate manipulation of hypersaline microbial mats reveals stasis in the cyanobacterial community structure. ISME J. 2:457-470

If you are interested in the manuscript, please contact me (sjg172@gmail.com) and I will send you a PDF. Alternatively, the article is available for free from the ISME Journal website.

Also, a very interesting article in this month's Applied and Environmental Microbiology regarding the effect of primer mismatches on overall PCR yield.
Bru et al. 2008. Quantification of the Detrimental Effect of a Single Primer-Template
Mismatch by Real-Time PCR Using the 16S rRNA Gene as an Example. AEM 74(5):1660-1663.

Cheers,
Stefan