RT PCR produces nice crisp band but of wrong length :-) - (May/15/2007 )
I would like to ask for an advice. I am trying to amplify a gene (about 800 bp) for a purpose of expression later on.
My idea was to extract the whole RNA, do RT PCR with nested primer (about 150 BP around my gene of interest). In the next step I wanted to use the cDNA obtained for one more PCR to amplify the gene of interest, which I would then later subclone to an expression vector.
The problem is that after RT PCR, for which I use outer nested primers, I get a band of 500 BP instead of expected 1100. The primers seem to be unique enough to not to amplify some other sequence.
I will greatly appreciate your suggestions.
did u check that your prediction of the PCR product was done on the mRNA sequence and not the genomic DNA, which may explain why you expect a bigger product than what you actually get.
If your prediction contained the introns, this may explain why it's shorter once amplified...
The probes were designed from mRNA and there shouldnt be any DNA contamination in the sample. Thanks