cDNA as PCR template - (May/21/2010 )
I have some basic doubts.
1. can cDNA (single strand) synthesized by the reverse transcription of total RNA be used as template for PCR?
2. If so how do we determine the concentration to be used?
3. How to make sure that our desired template is in enough quantity to be amplified in a degenerate PCR?
These things are way over my head.
Someone kindly help me out
1. of course
2. depends on how abundant that gene is and on your PCR conditions, just start with 1 - 2 ul of cDNA in RT-reaction per one PCR tube, don't care about concentration
3. you could try to amplify (or even quantify) cDNA of some similary expressed gene to be sure you have good quality cDNA