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
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'
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