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FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY
• In the primer extension step the polymerase at its optimum temperature
of72°C extends the primer sequence by synthesizing a new DNA strand
that is complementary to each template molecule, thereby doubling the
copy number of the target region. The PCR cycle is then repeated 25 to 40
times, thus leading to exponential accumulation of the PCR product.
The size of the PCR product is defined by the distance between the 5' ends of
the two primer annealing sites (Figure IAa). Therefore, the PCR product can
be visualized as a distinct band on agarase gel electrophoresis.
No single PCR protocol will be appropriate for allexperimental purposes.
Therefore, in the following a standard PCR protocol is given with subsequent discussion of the optimization of individual parameters for specific
applications. Additional information on PCR procedures can be found in
books on PCR methodology (1, 2).
Materials
Reagents
• DNA template
• Oligonucleotide primer pair synthesized on a oligonucleotide synthesizer
• Taq polymerase (e.g. Perkin Elmer Cetus)
• dNTPs(dATP,dCTP,dGTP,dTTP)
• 10 x reaction buffer for Taq polymerase (e.g. Perkin Elmer Cetus: 1 x
buffer is Tris-HC150 mM (pH8.3), MgClz 1.5 mM, KC125 mM, Tween 20
0.05o/o, autoclaved gelatin or nuclease free bovine serum albumin 100 )lgl
ml)
Solutions
• Ultrapure H 2 0
• Mineral oil
Supplies • Automated temperature cycling machine
• 500 ).11 Eppendorf tubes or heat resistant microtiter plate, fitting to the
machine
• Gel electrophoresis apparatus to analyse the PCR product
FRIEDHELM HILDEBRANDT AND IVA SINGH-SAWHNEY
• In the primer extension step the polymerase at its optimum temperature
of72°C extends the primer sequence by synthesizing a new DNA strand
that is complementary to each template molecule, thereby doubling the
copy number of the target region. The PCR cycle is then repeated 25 to 40
times, thus leading to exponential accumulation of the PCR product.
The size of the PCR product is defined by the distance between the 5' ends of
the two primer annealing sites (Figure IAa). Therefore, the PCR product can
be visualized as a distinct band on agarase gel electrophoresis.
No single PCR protocol will be appropriate for allexperimental purposes.
Therefore, in the following a standard PCR protocol is given with subsequent discussion of the optimization of individual parameters for specific
applications. Additional information on PCR procedures can be found in
books on PCR methodology (1, 2).
Materials
Reagents
• DNA template
• Oligonucleotide primer pair synthesized on a oligonucleotide synthesizer
• Taq polymerase (e.g. Perkin Elmer Cetus)
• dNTPs(dATP,dCTP,dGTP,dTTP)
• 10 x reaction buffer for Taq polymerase (e.g. Perkin Elmer Cetus: 1 x
buffer is Tris-HC150 mM (pH8.3), MgClz 1.5 mM, KC125 mM, Tween 20
0.05o/o, autoclaved gelatin or nuclease free bovine serum albumin 100 )lgl
ml)
Solutions
• Ultrapure H 2 0
• Mineral oil
Supplies • Automated temperature cycling machine
• 500 ).11 Eppendorf tubes or heat resistant microtiter plate, fitting to the
machine
• Gel electrophoresis apparatus to analyse the PCR product