Hi there,
Ive often asked myself the same questions. When PCRing 200bp
from a cosmid Ive found that less template gives rise to a better
amplimer yield. You could speculate that if you swamp the reaction
with template PCR priming will occur preferentially with the initial
template. You want this to occur in the initial stages but not in
later stages when you want exponential amplification. The initial
template maybe too complex to provide efficient amplification later
on. I find that for genomic clones 1ng DNA is sufficient and for
genomic DNA approx. 30ng (in 12ul) works fine.
cheers
Nick.
