PCR from cDNA library - (Jun/01/2008 )
I am trying to pcr from cDNA library, how much cDNA library should i use as template and what are the precautions i must take to get successful PCR amplification.
Information regarding the cDNA library size and average insert size may be available. From there calculate the number of plasmid copies you need to ensure that one copy of your gene would be present. I'd try to use about 100 copies of your gene, if you want to see a band after a single amplification (say 35 cycles per reaction). 10 copies if you plan to do a second nested amplification.
Or just try 10ng and 100ng of the library, and do a second round of amplification (using 1/10th of first reaction and overlapping primers), if you don't see the band the first time.