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.

Pourcentage gel d'acrylamide en fonction de la taille

Recommended Aery	amide Gel
Percentages for Resolution of Protein
Gel percentage	DNA size range
8%	40-200 kD
10%	21-100 kD
12%	10-40 kD

Pourcentage gel d'agarose en fonction de la taille

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Recommended Agarose Gel Percentages
for Resolution of Li	near DNA
Gel percentage	DNA size range
0.5%	1,000-30,000 bp
0.7%	800-12,000 bp
1.0%	500-10,000 bp
1.2%	400-7,000 bp
1.5%	200-3,000 bp
2.0%	50-2,000 bp