Optimisation PCR

PCR Primer Design Guidelines

Length : 18 to 30 nucleotides
GC Content: 40% to 60%
Annealing: For annealing temperature, use 5°C to 10°C below melting temperature (Tm).
Temperature: Example Tm calculation :
Tm (°C) = 2( #A + #T ) + 4( #G + #C )
Generally, a Tm between 55°C and 80°C will yield the best results.

Sequence: Avoid polybase sequences (3 or more) Gs and Cs at the 3' end.
Avoid mismatches at the 3' end. +
Avoid complementary sequences within the primer. +
Avoid primer-dimer formation by complementarity at 3' ends of primer pairs.


PCR Reaction Condition Guidelines


Primer concentration : - Final primer concentration should be 100 - 500 nM, which is equivalent to -100 to 250 ng of an 18- to 25-mer oligonucleotide primer in a 100-ul reaction volume. +
- Primers should be salt-free and gel-purified.