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

Dickens Lum lumdicks at netvigator.com
Fri Aug 8 10:14:39 EST 1997


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..

Regards,
Lum





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