Professional Documents
Culture Documents
Primer Optimization
Primer Optimization
Primer Optimization
50 mM KCl
1.5 mM MgCl2
The standard polymerase buffer works well for a wide range of templates and
primers but may not be optimal for any particular combination. Especially the
concentration of Mg2+ ions is critical and should be optimized. A series of PCR
experiments should be carried out with Mg 2+ concentrations varying from 1.5 to 4
mM in 0.5-mM steps. High concentrations of chelating agents (such as EDTA) and
negatively charged ionic groups (such as phosphates) should be avoided. Some
suppliers of DNA polymerases have added NH 4+ ions to their buffers. It has been
shown that the presence of NH4+ ions results in a high specificity of the primertemplate binding over a broad temperature range.
GC content of DNA template. PCR with GC-rich templates(>60%) are
especially difficult. This is mainly caused by the formation of stable secondary
structures that stall or reduce the polymerase reaction. Good results have been
obtained by the addition of glycerol, DMSO (5-20%), formamide (5-20%)
or tetramethylammonium chloride (0.01-10 mM) to the reaction mix.
Although little is known of the exact role of these chemicals in PCR. A commercial
additive is Q-Solution from Qiagen.
Inhibitory concentration
SDS
>0.005% (w/v)
Phenol
>0.2% (v/v)
ethanol
>1% (v/v)
>5 mM
sodium chloride
>25 mM
EDTA
>0.5 mM