How to remove DNA from mRNA...???

Keith James k.james at bangor.ac.uk
Sat Aug 9 07:06:50 EST 1997


Dickens Lum <lumdicks at netvigator.com> writes:

> 
> Hi Netters,
> 
>         I'm now trying to amplify a gene by RT-PCR. I've isolated the
> total RNA and then the mRNA by the mRNA isolation kit from Boehringer
> Mannheim. I then use AMV reverse transcriptase to synthesize first
> strand cDNA and use the product in PCR. However, as the target gene has
> numerous (>40 copies) pseudogenes ( with the same size of mRNA) in the
> genome, I'm not sure whether the product is amplified from the DNA or
> mRNA-cDNA hybrid. In fact, the negative control (using the isolated mRNA
> as template) also give a PCR product of the same size. As those
> pseudogenes contains frame-shift mutation or have pre-mature stop codon,
> they can not be used in my expression experiment.
> 
>         My question is: how can I get rid of the DNA in my mRNA
> preparation...???
> 
> Any comment and suggestion are welcome. Thanks in advance..
> 

I'm about to use RT-PCR to demonstrate induction of an operon. This puts
me in a similar position of having to avoid amplification of DNA from
the total RNA prep.

The route I'll be using is to treat the RNA prep with DNAse prior to
amplification. I've ordered some RNase-free DNAse to do it. I haven't
tried this yet myself, but others in our lab have used it successfully
on prokaryotic RNA preps.

-- 
Keith James Ph.D. - k.james at bangor.ac.uk  PGP 2.6.2i  Key ID 469A9FA1
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