Factors to be considered for primer optimization:

Primer concentration. The concentration of each primer should be between

0.1 and 0.5 M. For most applications 0.2 M produces satisfactory results. Too
high primer concentrations increase the chance of mispriming, which results in
nonspecific PCR products. Limiting primer concentrations result in extremely
inefficient PCR reactions. The primer concentration can be calculated as
described inPreparation of oligo solutions.
DNA template concentration. The concentration of DNA template depends on
the source. Normally used concentration are 100-250 ng for mammalian genomic
DNA and 20 ng for linearized plasmid DNA (circular plasmid DNA is slightly less
efficiently amplified) per 50l reaction.
Concentration of dNTPs. The concentration of each dNTPs (dATP, dCTP, dGTP
and dTTP) should be 200 M. Too high concentrations of dNTPs inhibit the PCR
reaction.
DNA polymerase. The PCR reaction conditions and reaction times depend on
the type of DNA polymerase used. Different polymerases are commercially
available and are summarized in DNA polymerases.
Polymerase buffer. All DNA polymerases are supplied with their own optimal
polymerase buffer.
Standard Polymerase buffer:

10 mM Tris-HCl pH 8.3 (at room temperature)

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.

Annealing temperature. The optimal annealing temperature has to be

determined experimentally. As a starting point, an annealing temperature 5C
below the estimated melting temperature (Tm) can be used.
Cycle hold times. The optimal hold times for the denaturation and annealing
steps in each cycle depend strongly on the design of the thermocycler and the
wall thickness of the PCR tubes used and should be determined experimentally.
The hold time for the extension step depends mainly on the length of the DNA
template. As a rule of thumb we use 60 sec per kb. Ramp times are normally
kept as short as possible.
Number of cycles. The number of cycles necessary to obtain a suffient amount
of PCR product depends strongly on the concentration of the DNA template. In a
typical PCR, the maximum amout of product is approx. 10 12 copies of the
template. Starting from one copy, the most efficient PCR would reach this level in
40 cycles. Depending on the nature of the DNA template, we start with many
more copies and as a rule of thumb we carry out PCR with 25 cycles for plasmid
DNA and 30-35 cycles for genomic DNA.
Inhibition of PCR by impurities
Impurities introduced by the Plasmid Mini/Maxi Prep and/or DNA Purification kits
can have strong inhibitory effects on PCR (see table below).
Substance

Inhibitory concentration

SDS

>0.005% (w/v)

Phenol

>0.2% (v/v)

ethanol

>1% (v/v)

isopropanol >1% (v/v)

sodium acetate

>5 mM

sodium chloride

>25 mM

EDTA

>0.5 mM

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cr/index.html