qRT-PCR and DNA contamination

Brian P Higgins via methods%40net.bio.net (by higginsb78 from gmail.com)
Thu May 22 12:10:50 EST 2008


Have you tried swapping out ALL of your reagents?  Many times trace DNA can
be found there.  Do you use barrier tips for every single procedure in lab,
or just your RNA work?  Hopefully you have RNA only pipettes, but if not,
you can try wiping down the barrels of the pipettes to get rid of any
contaminating DNA (it's a bit extreme, but why not cover everything :)  Make
sure all of your reagents are used for RNA work only as well, as someone
else in lab could be contaminating them if they are for general use.  Sorry
if you've already done all these--they are the basic steps to getting rid of
DNA, but your email didn't stipulate what measures other than DNase you've
taken.

Also, are you detecting the DNA contamination on a gel or in the qPCR?  If
the latter, what cycle?  If it's anything past 28-30 cycles, check the melt
cure to make sure it's truly contamination--most negative controls will
accumulate enough junk at cycle 30 or so to give you a weak Ct.

Good luck,
Brian

On Thu, May 22, 2008 at 5:17 AM, Emad <e_barouki from hotmail.com> wrote:

> 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
>
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