DGGE Help Page

I am announcing the release of my DGGE Help Guide. I have spent some time reviewing the questions that have accumulated on the YahooGroups DGGE page and decided to compile a guide to answer many of the questions that come up repeatedly. The guide is not meant to be definitive, but it is meant to be a resource for users to find the information that they do need. I also put in some of my own personal opinions regarding PCR and DGGE.

I hope that people find the guide useful. I will update the guide as time goes along, and I will be happy to receive additional information from anyone interested in providing it. If anyone feels that there are sections that they would like to write, update or correct, please contact me.

I have the guide on another blog: http://ddgehelp.blogspot.com/
The entire text can be had in word format or PDF format. However, I have found that the PDF format does not retain the hyperlinks. For the intended format, please use the word file.

Monthly Update

Well, it seems about time for a monthly update. I had thought I might do this more routinely, but I guess this is what we can expect! I have added a few tidbits to the SuPER PCR page. These include citations to two very nice manuscripts. One is a method similar to SuPER PCR but for RNA and the other is a clever mechanism to circumvent the problem of plastid DNA in environmental studies of plant roots.

Things here are going well. I am in the midst of a major sequencing effort relating to the "Sulfate II" experiment that was conducted at NASA. The idea was to take hypersaline microbial mats (See Bebout et al. 2005; Minz et al. 1999 and many others) and incubate them under controlled conditions at standard salinity (85 ppt), lowered salinity (35 ppt), at standard sulfate concentrations (~60 mM) and at low sulfate concentrations (< 1 mM). After letting these mats acclimate to these conditions, a suite of geochemical measurements were taken (oxygen/sulfide profiles, methane evolution, etc), and I have been working, together with Jason Smith (U of Florida), Mary Hogan (NASA/ARC), Brad Bebout (NASA/ARC) and Lee Bebout (NASA/ARC), on analyzing the methanogens and sulfate reducers in the system. The preliminary analyses of the methanogens are quite striking and indicate a dramatic shift in population as a result of the removal of sulfate. More on that when all the data are in. No sequence data yet on the sulfate reducers.

I have also been performing some phylogenetic analyses with the program Mr.Bayes (discussed in a prior posting). However, I am having problems with the technique - with regard to the branch lengths that the method is yielding. I have noticed that the branchlengths produced by MrBayes are significantly longer than those I get from other treeing methods such as Neighbor Joining or Maximum Likelihood. I have observed that the branch lengths are roughly 2x those of NJ, and 2x thes equence difference (as estimated by BLAST or other aligning method). I realize that MrBayes branch lengths are "expected changes per site" and not necessarily the same as NJ branch lengths. Still, the 2x factor seems excessive. If anybody has any thoughts on this I would love to hear them. Overall, the branching order yielded by Mr. Bayes seems quite consistent with other methods.

Well, more soon.