Polymerase Choice Troubles - my primer melting temp is so low (~30 C) (Jan/29/2007 )
I am having trouble amplifying some cDNA that I made via reverse transcription. The primer melting temperatures for are quite low, between 27 and 33 degrees C, and I think (hope) this is causing annealing problems. I am having trouble finding a proper polymerase to carry out my reaction; does anybody have a good choice to share?
I need to try some suitable polymerase before I'm willing to consider that maybe the reverse transcription didn't go well. I have no immediate means to test whether it did or not anyway.
are you really sure of your primers??????
For getting 30°, only a 10bases primer would fit, which seems very few for accurate RT.
Yea, I'm sure it's that low. The one primer reads TTTTTTTTTTTTTTTVN; it's a 17 bp primer with a Tm of 26 degrees at the low end. Maybe just add more T's to the primer for RT? But then I lose a lot of the mRNA with shorter poly A tails.
wow, you don't want to use the cDNA synthesis primers to make more cDNA via PCR...there's a lot of room for error here
is it possible to use gene-specific primers for the amplification of the cDNA? or, start with more RNA and synthesize a larger amount of cDNA? for which application will you use your cDNA?
I should clarify something. I using the poly A primer in PCR would have been paired with a gene specific primer. The aim would have been to amplify the sequence 3' to the GSP. This is what I'm worried about.
Also, does anyone have any recommendations on using poly T as a primer for reverse transcription? Maybe this is no the right thread/forum for this question, but I am having difficulties carrying out RT on samples with a 42 degree elongation temp. Please advise me if any of you have an idea.
Also, if using the poly A primer with the GSP in a PCR with RT template is a bad idea, please let me konw why.