In theory yes, but in reality no.You want to use rT-PRC with the mRNA as template to produce cDNA. Unfortunately, if you do colony PCR you will have DNA and RNA. If you use DNase to digest your DNA, this DNase will digest your cDNA, primers etc...If you dont do DNase digestion, your PCR will be mainly on the DNA, amplifying genomic amplicon (or your plasmid). The best would be colony lysis, DNase digestion, purification and then rT-PCR...And the only logic way to do it actually. If, on the other hand, want to work on DNA to amplify your target gene, then it is possible.
In theory yes, but in reality no.You want to use rT-PRC with the mRNA as template to produce cDNA. Unfortunately, if you do colony PCR you will have DNA and RNA. If you use DNase to digest your DNA, this DNase will digest your cDNA, primers etc...If you dont do DNase digestion, your PCR will be mainly on the DNA, amplifying genomic amplicon (or your plasmid). The best would be colony lysis, DNase digestion, purification and then rT-PCR...And the only logic way to do it actually. If, on the other hand, want to work on DNA to amplify your target gene, then it is possible.
DNA contamination will remain a problem and you can not avoid it using direct colony. for cDNA (complementary DNA prepared from expressed mRNA) pure mRNA without DNA contamination is must. Else u will end up with amplification of both DNA and cDNA (mRNA) and you cannot get the actual expression profiles for a given gene.
Colony lysate treated with DNase could be used but stability of mRNA would be an issue.
Thanks Mr Dogra for your suggestion..! But, I have a confusion as you mentioned that there is possibility of DNA amplification along with cDNA. Actually, I don't know if reverse transcriptase can amplify DNA also! Is it the case?
Dear Anjan
Its not the Reverse Transcriptase that will amplify your DNA (It can't).
DNA will remain there untouched in your RT reaction but as soon as you will go for amplification of your desired gene, off course with a DNA polymerase, your DNA will give amplification.
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