I need some help with a PCR. I am amplifying cDNA that was converted from RNA of a tissue section. I have three donors. I am targeting a specific gene and used different annealing temperatures.
The first time I carried out the PCR, everything worked at one of the annealing temperatures. I got the right bands, then cloned, grew colonies on agar plates, and Sanger sequenced for verification of target and it was verified.
I repeated the cloning and agar plate colony growth to get more colonies. The second time I didn't see the same PCR bands at the same annealing temperature. Instead I saw the correct bands at different annealing temperatures. I thought it was strange, but I cleaned the product up (PCR purified) and again streaked agar plates for colonies and Sanger sequencing. I saw colonies on all the plates, colony picked them by restreaking on a new agar plate, then submitted them out for Sanger sequencing. The vendor said there was no colonies that grew.
My question are
-what protocol do you recommend for cDNA amplification of gene targets to detect isoforms? Do I have to plate them for colony picking after PCR? Is it necessary to do a PCR cleanup?
-My PCR bands were very faint both times, what polymerases do you recommend for this type of workflow?
-what part of the steps I wrote above would you troubleshoot?