Wednesday, December 20, 2006

Some more publications of interest and silver staining

- Immediate link to DGGE Guide -

* Please notify me if there are links that are no longer valid. - Thanks, Stefan

Some newer publications of potential interest to the DGGE community:

A double-gradient (acrylamide and denaturant):
Analysis of Bacterial Communities in Seagrass Bed Sediments by Double-Gradient Denaturing Gradient Gel Electrophoresis of PCR-Amplified 16S rRNA Genes
. Microbial Ecology: Volume 52, Number 4 / November, 2006. J. B. James, T. D. Sherman and R. Devereux.

A new approach combining both sequencing and DGGE in a clever manner:
Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries
. Microbial Ecology: Volume 51, Number 4 / May, 2006. M. D. Burr, S. J. Clark, C. R. Spear and A. K. Camper.

In addition, I have had a very nice correspondance with Ing. Oscar de Vos from the Biological Farming Systems group at the Wageningen University. Oscar wrote me to inform me that silver staining is still being used. He stated:

"I would like to mention that I still use silver staining (oxidizer, silver nitrate and developer from Bio-Rad) to retrieve nice pictures of our soil/manure samples with about 100 to 150 bands per sample. I once verified our Phoretix software; does the software detect bands that I can detect by eye? Using a vertical illuminator with my gel attached to it I found sometimimes 3 distinct bands per mm. Nice thing about using silver in combination with Gelbond (Amersham Biosciences) is that you can cover it after preservation with cellophane sheet (also from Amersham) and dry it at room temperature for 3 days. Result is a well-preserved gel with an example in the attachment."

As you see, the diversity detected in these samples is tremendously high, and this raises questions about our capacity to observe shifts in microbial communities and to compare levels of diversity. I inquired how Oscar dealt with these issues, and he responded:

"I added a very recent correspondence with the technical support from Nonlinear Dynamics (Phoretix, TotalLab). I was having a bad time comparing lanes across gels, even comparing lanes within a gel is hard with such a high diversity. We are looking for alternative ways, but until then we just use species diversity/richness, which is a valuable parmeter for out research (in the added mails, you may find a brief outline of our research). Zooming in with DGGE for interesting regions is an option, but we are very much interested in the whole eubacterial community for example of manures derived from cows with different feeding regimes and relate this to the survival of the human pathogen E. coli O157:H7 in this manure. It is nice that you have seen the difficulties in this research.


Arjen Speksnijder was in the lab lately and liked the sharp bands and diversity in the gels I sent you very much, it is quite a difference with the sometimes fuzzy bands he retrieves with Ethidium Bromide, but with GelCompar and his low diversity gels he has the advantage of having a good comparison across gels..."

I think the article by Burr et al. 2006 (Microbial Ecology), may help resolve some of these issues by making sequencing/DGGE more of an integrated approach. I highly recommend looking at it as a way to help overcome some of these difficult issues in characterizing communities with high levels of diversity.

Happy holidays to all.
Cheers,
Stefan

51 Comments:

At 7:05 PM, Anonymous Anonymous said...

Hi, I was wondering if you have any ideas as to why there is an area at the top of the gel that does not seem to be staining. I have been having this problem using the C.B.S DGGE system recently, but did not have it before. I am using SybGold to stain and Oscar's gel was stained with silver nitrate and we have the same problem. Any ideas?

 
At 2:16 PM, Blogger Stefan J. Green said...

I have seen this in many gels from different systems and different dyes, and I must admit I have never been able to identify the problem specifically. I suspect that it does not have anything to do with the type of dye used, but rather is some artifact of the pouring of the gel. In my previous lab we had some users who always got this "no DNA" zone while others did not have it. Since it tends to be very high in the gel, I have never worried much about it. However, perhaps other users may have a better idea as to the source of this, and ways to avoid it.

Some possible sources / solutions:

Temperature at time of pouring? Allowing the gradient to polymerize before adding the overlying supernatant?
Extra APS and TEMED in the supernatant (may prevent)?

Cheers, Stefan

 
At 7:02 PM, Anonymous Ana said...

Thank you Stefan. We run another gel today using the DeCode system and we had the same problem, so I don't think is the system either. Our latest theory is that it is something in the solutions, maybe a bad batch of urea since it always appears in the denaturing part of the gel and no in the stacking gel (we run some gels without stacking gel and the same thing happened). The reason we were wondering is that after the "no DNA" zone we get very dark smears even after cleaning up the PCR products and it seems to affect some of the bands running properly. Will try new reagents and let you know.

 
At 3:31 AM, Anonymous Anonymous said...

HI, I was wondering do you know if the GC clamp in the primers somehow affects the negative in the ordinary PCR reaction? I have tried everything from changing the reagents, changing the water and also changing the amount of cycles in the PCR run but still to no avail. when I run the PCR products on an agrose gel the negative shoes up.

 
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At 11:54 AM, Anonymous Anonymous said...

Hi,

Love this blog, very helpful as I work through all the issues related to running DGGE.

I have a couple solutions for those that have posed questions above.

You can get rid of the blank area at the top of the gel by running at a lower voltage (~50V) for 15 minutes prior to increasing to say, 100V for 10 Hrs. Every time I run a gel with an immediate start at 60V or higher, I get a blank area at the top of the gel.

Also, I had a lot of problems with contamination in my negative controls as well. I was using universal bacterial primers with sensitive touchdown PCR conditions. I found two things. Often the taq is contaminated from the company, thus I UV irradiate the taq and water prior to loading primers and template DNA. Also, as you hinted at, -GC clamped primers tend to be contaminated with small bits of DNA unless you buy them cleaned up (e.g. HPLC). Try buying cleaner (more expensive) primers first, and if you still have contaminants, try irradiating your taq and water for 20 min prior to use.

Good luck!

 
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