qRT-PCR and DNA contamination

Sudheendra Rao N R via methods%40net.bio.net (by sudhee26 from gmail.com)
Thu May 22 11:47:55 EST 2008


Hey,
i have never isolated RNA using columns..but just Trizol and rarely i get
DNA contamination.
It is surprising that DNAse still not able to digest your DNA
i suggest u to run following control lanes in the gel
1. just DNA loading Dye
2. control DNA incubated with DNAse
3. Just DNAse

i guess that must trouble shoot it.

Sudheendra.

On Thu, May 22, 2008 at 2:47 PM, 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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