PCR on cDNA libraries - (May/18/2001 )
I want to amplify cDNA libraries using a gene specific primer and the phage vector primer.Has anybody got an optimised protocol for this.
Hi!if you have both specific primers, the task is simple. just your expected annealing temp (try 1 or 2 degrees below),should work. I generally take 20 ul of the library, boil it for 5 min, snap chill and use it as template forPCR. If you have either forward primer, and if your librarary is ZAP, you could tag it with T3 or T7 primers. T3 or T7 amplify around 55C, so you could design your specific primer around that temp.
I have a question to you, how to design a primer which works at around 55, any program you can suggest or just an advice to do it manually.