qRT-PCR and DNA contamination

Emad via methods%40net.bio.net (by e_barouki from hotmail.com)
Thu May 22 04:17:07 EST 2008


Hello,

I am doing real time PCR for analysing my genes expression under different
condition of my fungus. We use SYPR green. Actually I isolated RNA using
both kits Qiagen and Peqlab (Germany) and treated the RNA with DNase on
column following the kit's instruction. Unfortunately I could still detect a
DNA contamination!! I treated the RNA again with DNase from NEB but still
some traces of DNA exist. I tried to avoid the DNase treatment because I
learned from some people who deal with RT-PCR to avoid DNase treatment and
used Intron Span primers, and here is the big suffering, I used 18-20mere
primers which locate just in the middle of exon borders and still these
primers can bind the pure DNA, I reduced to 6 bases on the 3'-exon and still
get some amplification, isn't that terrible? I thought to go to 3 bases at
the 3' end of the primer but then you don't get any good prime which suit in
this area!!? 

Can somebody please give me some hints or has any experience in such a good
DNase which doesn't leave any DNA traces?! I am not optimistic in that. 

Emad 



More information about the Methods mailing list