PCR from genomic DNA - (Jul/18/2005 )
....I have a hard time, trying to get promotorfragments from human genomic DNA by simple PCR....first I used Primers with RE-sites which do not bind 100% correctly.....didnt work at all....after that I wanted to do some nested PCR with more simple primers, generated by primer3, best conditions (Tm 60 °C) and no misspriming.
and I get.......unspecific product or nothing.....
...can anybody help me with some good hints, please
What is your template concentration? Have you tried altering the DNA concentration in your reaction mix?
If I do PCR with genomic (bacterial) DNA, I usually dilute my sample 1:50 and use 5-10 ul.