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YOSHIO KANEMITSU AND TAKEHIKO KOJI
1. Mix the following in PCR tube on ice:
Reverse transcription product
or cDNA (10- 18 _10- 15 mol)
PCR buffer (Mg 2 + free)
50 mM MgC12
10 11M sense primer
10 11M antisense primer
5 mM dNTP mix
Taq polymerase
DDW
10 III
3.4 111* 14.0 III
5 III
5 III
4 III
0.5 III (=2.5 U)
up to 100 III
2. Set the sample tubes on the thermal controller and perform
the following steps:
1 cycle:
94°C
1 mins
30 -40 cycles:
94°C
40 secs (denaturation)
55°C
40 secs (annealing)
noc
40 secs (extension)
1 cycle:
noc
3 mins
4°C
continuous
Annealing temperature must be changed according to the
melting temperature of the primers. The optimum time
parameter may differ in each case on account of the performance of the thermal controller, the volume of the solution
or the sequence length to be amplified.
3. Check the size of the PCR products by agarose gel electrophoresis.
4. Remove the mineral oil. Add equal volume of buffered phenol and vortex briefly. Centrifuge 5 mins at 5000 rpm and
save upper aqueous phase.