preferential amplification among transcripts - (Feb/07/2006 )

I am doing PCR on cDNA then cloning and sequencing.

So far I have only managed to find one version of the transcript though I know there are two (and I would like to amplify them both).

The transcript I am amplifying is 230bp and the other is 280bp. So, Question 1: Is this enough of a difference for the PCR to amplify one in preference to the other? Or will the bacterial cells preferentially take in a shorter transcript?

Question 2 Is it possible for primer annealing to be influenced by sequence outside of the 20 bases? The end of my primer is only 4 bases distance from the splice site (at which point the two transcripts would differ). Is it possible this could cause a PCR bias?

It's not a major problem just curiosity.

i wouldn't have thought that 50bp would make any difference in the context of the ?kb long vector....
maybe your primer for the second transcript isn't very efficient?
Are you sure that both transcripts are expressed in tissue/cell line from which you extracted the RNA? Maybe try another tissue?
you could try nesting the PCR or using a specific primer to make the cDNA (rather than oligodT or (N)6 )?

finally - maybe you just need to check heaps of colonies!