Thursday, October 16, 2008

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."

6 Comments:

At 10:34 AM, Anonymous canalon said...

Hi I stumble upon your blog as I am starting to implement DGGE in my lab. I have compared TAE and SB buffer and have settled on TAE as I got much better separation with this buffer.

I would send pictures, but I did not find your e-mail.

Cheers

 
At 1:40 PM, Blogger Stefan J. Green said...

You can send emails to sjgreen@fsu.edu

Well, too bad about SB buffer for DGGE....

 
At 4:37 AM, Blogger Michał said...

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.

 
At 11:20 AM, Blogger Watson said...

Yep, that's true.
I also had great expectations with SB buffer. It didn't work out. Gels were soft, fragile and they significantly expanded their volume when I was washing out urea/formamide. Additionally, the smell of ammonium was present. It was almost impossible to transfer them into visualisation chamber. There were no sharp bands, only blurry smeared stains.
It seems that we have to stick to TAE. Maybe LB buffer? I'll try this next time.

 
At 11:21 AM, Blogger Watson said...

Crap, It seems that I replied to my own comment... :)

 
At 8:04 PM, Anonymous Panic Attack said...

I had a similar experience as Michal and Watson regarding the SB buffer. What solution would your recommend? Are there other alternatives?

 

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