PCR Optimization: Reaction Conditions and Components

Consideration for components of the PCR optimization:

Primer Design: Oligonucleotides used for priming the PCR should be at least 16 nucleotides and preferably 18-25 nucleotides in length. They should contain 40% -60% G+C. Avoid sequences which would produce internal secondary structures. The 3’-end of the primers should not be complementary to avoid the production of primer-dimers in the PCR reaction. Avoid three G or C nucleotides in a row near the 3’-end of the primer. Ideally, both primers should anneal at the same temperature, differ not from 5 ?C. The annealing temperature is dependent upon the primer with the lowest melting Tm.

Buffers used for PCR: The standard buffer for PCR contains 50 mM KCl, 10 mM Tris.Cl (pH 8.3 at room temperature), and 1.5mM MgCl2.

Enzyme concentration: 1.25 units of Taq DNA Polymerase. Pipetting errors are the most frequent cause of excessive enzyme levels. The use of reaction master mixes is strongly recommended.

Deoxyribonucleoside triphosphates: dNTPs mix (10mM of each dNTP)

DMSO: About 2.5% can increase product specificity.

Template: 10-100ng for 50 ?l final reaction.

Water: Distilled water which has been filter sterilized or autoclaved.

Last update 29-Oct-2001, Rating Good of 3 votes.