DGGE - DNA migration

[TomMol]

Hello,

Two months ago I started use DGGE method. I want to use it to detection bacteria (mainly Lactobacillus) in stool. I have got problem. When I put PCR product into wells samples didn’t migrate equally. It migrate faster on both periphery gel and slower in center. I added 100 ul 10% APS and 10 ul TEMED per 25 ml poliacrylamide. I used 100 V and temperature 60C – 10 % gel.

What should I change in my procedure to my results will be better?
Thank you

Tom

tgosiews@cm-uj.krakow.pl

[Niqqie]

Either it is a problem in the way the electricity is flowing through your gel, or it is a problem of how the gel polymerizes. I personally think 100V is a lot, I run 7% polyacrylamide gels at 60V; I only use a high voltage to get the samples into the gel, after which I turn it down.