DNA contanmination in RNA prep

Richard P. Grant rgrant at netscape.net
Mon Jul 10 02:41:48 EST 2000


In article <3965E9D0.E3DE860C at ikp.unibe.ch>, "Lu Z. H." 
<zen.lu at ikp.unibe.ch> wrote:

> I then treated the RNA with DNase I. However, my
> no RT control kept giving me positive signal in my PCR. Thank you!

If your DNase is still alive (and you have used the correct amount), 
then we could assume that you have no or non-significant amounts of DNA 
in your prep.

Therefore, it is likely that you have DNA contamination else where.  
Likely targets are the water and the PCR buffer.  Solution?  Throw out 
all working aliquots and use fresh reagents.  Don't bother trying to 
find out which is contaminated, life is too short for that kind of thing.

If you're not convinced, use the same primers but perform the RT-PCR on 
a well-characterized piece of cDNA [sic] (pBluescript, for e.g.) that 
does not have the PCR target - if you keep everything else trhe same but 
still get a band, then you know there is template contaminating 
something.

HTH

Richard

PS Using that method of RNA preparation, I used to find that 
DNase-treatment was superfluous anyway.

-- 
Richard P. Grant MAD Phil        http://www.gerbil.org.uk/    
Please reply to rpg 'at' mrc-lmb.cam.ac.uk'

         Sex and bugs and rock and roll






More information about the Methods mailing list