pcr-rflp - (Jul/21/2010 )

Hi

I need to digest a 164 bp PCR product to detect a gain-of-res-site point mutation. I am using SfcI.The protocol I follow (primers, annealing temp, reaction conditions) are well established.

However, I have run into problems in digestion. I tried 5 hrs, overnight at 37C, even changed the rxn vol from 20 ul to 10 ul, vol of PCR prod from 10ul to 8 ul. However, all I get is a smear whereas I need to get 2 bands of 109 and 55 bp.

My gel is 2 % agarose (should I increase this ?). Also, pls let me know a good protocol for silver staining of polyacrylamide gels for DNA.

I welcome your suggestions.

Thank you

Are you sure you can see clearly a 109bp and 55bp band on agarose gel? I really doubt that...
Perhaps you can try using PAGE gel.

Just my 2 cents.

I agree with Adrian about trying to distinguish such 109 bp bands from 55 bp and from anything that might be undigested on an agarose gel. Also, 5 hrs is a pretty long time for a restriction digest. What is the recommended time according to the enzyme manufacturere? I was trying to do something similar and found that if I overdigested my samples, I would get some random cutting.