We have tried out with 9% gel and 30-60 % ,40-60%denaturing gradient but still our bands are only visible lower side the gel.
why don't you try a wider gradient such as 20-70%? You may need a wider gradient to view more bands. Here is a blog I used when trouble shooting DGGE.
http://ddgehelp.blogspot.dk/2005/11/dgge-guide.html
Also did you check your PCR product to be good quality before DGGE?
if you are getting bands at lower end (high denaturation conc) in DGGE mean your amplicons are GC rich and the denaturing conditions are not sufficient to melt DNA. You need to increase the concentration of denaturant. you can try 40-80%. Also you should maintain 60 degree C temperature throught running the gel.
Thank you to all answer .I will apply this....
my Product size is 473bp.
For bacteria, we used 6% polyacrylamide gels with a 45% to 55% vertical denaturing gradient for DGGE.
Which size are your samples?
We usually amplify and run the V6-V8 region id est approximatively 450 bp in a 6% polyacrylamide gels 40-60% denaturing gradient.
Thank you all for your kind information.i got the result,i use 9% polyacrylamide gels 40-60% denaturing gradient.
thank you very much.
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