DNA migration in DGGE - (May/01/2005 )
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Hello,
Two months ago I started use DGGE method. I want to use it to detection bacteria (Lactobacillus mainly) from stool. I have got problem. When I put PCR product into wells samples didn’t migrate equally (like smile). It migrate faster on both periphery gel and slower in center. I added 100 ul 10% APS and 10 ul TEMED per 25 ml polyacrylamide. I used 100 V and temperature 60C – 10 % gel; denaturating gradient: 20% - 80%.
What should I change in my procedure to my results will be better?
Thank you
Ps. I used IngenyPhorU2 machine.
Tom
tgosiews@cm-uj.krakow.pl
Hello Tom...
I did has the same problem when the 1st time i use the technique. Have u did the perpendicular gel analysis? If not,then u should ran ran your PCR product 1st with perpendicular gel with suiteable gradient range based on the sized of PCR product. The results gained from perpendicular analysis will suggest u the optimal gradient ranged for your parallel dgge analysis.
Other tip to overcome the poor DNA migration is by having the best amplified PCR product (no smear, no multiple bands). So gud luck...
hi
i'm surprised because of the fact when i read infos about high MW dna markers, migration was done at 4°...
Hello,Two months ago I started use DGGE method. I want to use it to detection bacteria (Lactobacillus mainly) from stool. I have got problem. When I put PCR product into wells samples didn’t migrate equally (like smile). It migrate faster on both periphery gel and slower in center. I added 100 ul 10% APS and 10 ul TEMED per 25 ml polyacrylamide. I used 100 V and temperature 60C – 10 % gel; denaturating gradient: 20% - 80%.
What should I change in my procedure to my results will be better?
Thank you
Ps. I used IngenyPhorU2 machine.
Tom
tgosiews@cm-uj.krakow.pl
Hallo Tom,
look at Brinkhoff, T. and Van Hannen, E.J. 2001. Use of Silicone Grease to Avoid 'smiling Effect' in DGGE. Journal of Rapid Methods and Automation in Microbiology 9:259-261.
SMILING GELS
Smiling of bands near the edges of DGGE gels appears to be endemic in all systems. While the exact cause of this is not entirely clear, the smiling effect can be held in check by two approaches, best used together:
· Don’t load PCR product in the very far lanes.
· Apply grease to the spacers. Older spacers may require more grease than new spacers, but don’t overload the spacers with grease. A thin film is adequate.
Best
Sylwia