Strange frameshift question - (May/07/2010 )
My boss appears to have made a mistake back when we were doing the cloning of our insert into our vector and introduced a frameshift via cloning primer (which we seem to see from sequencing) from the ATG start codon. The strange thing is that when using primers which bind outside of the insert region, on the plasmid itself, on mRNA (Reversibly transcribed to cDNA) from cells transfected by this plasmid I see message. At first I thought I had genomic DNA contamination but my no RT controls are not showing any bands. Is it possible for a frameshift such as this to force the cell machinery to transcribe the ENTIRE plasmid as mRNA and not just the insert? Am I mistaken in believing that a frameshift of -1 base pair would only affect translation of the proteins?
Frameshifts have no effect on transcripton, only on translation.
Thanks Phage. Is it possible that I'm just amplifying from my plasmid that was transfected into the cells? I had thought that the dnase treatment would remove the majority of the genomic dna and plasmid but I was told that dnase will not digest circular plasmid. Only if it is knicked.
How does one distinguish the plasmid amplification from actual mRNA signal in a transient transfection?