poor polymerization in DGGE

Mark nospam at nspam.com
Tue May 10 07:39:23 EST 2005


Scott Coutts wrote:
> Rafael Maldonado wrote:
> 
> 
>>WU Yucheng wrote:
>>
>>
>>>Hi everyone,
>>>
>>>I am investigating the bacteria diversity in soil by DGGE method.But a
>>>poor polymerization always happened during casting. In fact,the
>>>acrylamide solution doesn't polymerize from below the comb about 1cm to
>>>the top even overnight at temperature(about 20¡æ). The gel oncentration
>>>is 8% and 10ul TEMED and 150ul 10%APS are added to 15ml high or low gel
>>>solution.
>>>
>>>Then,who can tell me what is the reason and how I can optimize the
>>>polymerization.
>>>
>>>Thank you in advance for your help!!
>>>
>>
>>Most of the time, poor polymerization is due to oxygen (from the air, if
>>the buffer is shaked) 
> 
> 
> Yes, it sounds like air. Oxygen inhibits polymerisation. Are you using a
> vertical gel system? It sounds like you need to seal the tops of your
> apparatus, if it is setting at the bottom but not at the top (where the
> oxygen gets in). What gel equipment are you using? If you're using a
> vertical gel, then sometimes you can push the comb further into the
> casting apparatus between the glass plates, so that the top is
> completely sealed off. We use the Biorad Mini-Protean II and III
> systems, and with the protean II systems we push the comb in further. By
> default, it will leave a little gap, about 1mm, above each 'tooth' of
> your gel (in between the wells). Air can get in the gaps and cause it to
> fail to polymerise. If you push it in further, it will work OK.
> Sometimes you need to bend the outside edges of the comb in a little
> towards the front of the gel so the comb doesnt rest on the top of the
> glass plate.
> 
> Cheers,
> 
> Scott.

That's a good suggestion. I have also seen people overlay the top
of vertical gels with distilled water, or water saturated
butanol. For the latter, just be sure to rinse it all off before
you do anything with the gel.

Mark



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