I was trying to use DGGE to separate different microbes. I used 8% Acrylamide/Bis-acrylamide gel with 10%-40% denaturant and ran 280 mins (130 V), and the DNA seemed to move into the gel a little bit. Therefore, I tried again to run longer for 840 mins (130V), and the DNA samples looked almost the same as the one I ran 280 mins. Each sample contains at least 450 ng of DNA. Did anyone experience a similar situation and know what's wrong with my experiment settings?
PHOTOS: The attached photos are the result of 280 mins and 840 mins, respectively. The sample on the right side is the 100 bp ladder, and the ones in the middle and left are DNA samples.
What's the length of your DNA fragments?
Looks like gel gradient 10-40% isn't ideal. For same 8% Acrylamide/Bis-acrylamide gel try increasing the gradient from 10 to 60 and apply very low voltage around 85V for at least 10--12 hours at 60 Celsius degrees.
Thanks for your suggestion!
The sequence length of my sample is about 1000 bp.
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