PCR primer-target annealing is a dynamic balance between primer-target association and dissociation,
the increase in primer and/or target concentration will facilitate the annealing, that's why there's a minimal primer concentration of
300 nM for the efficient PCR. Sometimes, if this minimal primer concentration is somehow impractical, the prolonged annealing time
can compensate for the deficiency. It's a trade off between primer concentration and PCR annealing time. Below is the demonstration of
the relationship between primer concentration and PCR annealing time.
Fig. 1000 copies of DNA templates were PCR amplified in 20uL,
with 300nM constant probe concentration and varying
forward and reverse primer concentrations. PCR annealing time varied from 0.3 minutes to 5 minutes.
Results revealed PCR primer concentration at at least 600 nM will guarantee an efficient PCR reaction regardless of annealing time,
primer concentration of lower than 600 nM would require longer PCR annealing time.
While you are here :-), introducing:
Conveniently Extract RNA!
Extract plasmids for quick PCR or sequencing screening.
Over 700bp CE sequencing readout directly from an overnight culture of a 14kb low copy plasmid