Taq Polymerase 43
- Temperature assessment in the heating block: (a) the time reading starts,
when the desired value in the block is reached or (b) the time reading
starts independently of the effective temperature of the block; - Temperature assessment in a reference vial: the time reading starts, when
the rated temperature in the reference is reached; and - Temperature assessment in water baths: the time reading is indepen-
dent of the actual sample temperature.
Different from the programmed temperature profile of the heating
block, the actual profile in the reaction vial is distinctly more shallow,
since the heat transfer through the material of the vessel is fairly bad.
For a successful PCR experiment, a set of defined temperatures
must be adjusted:
- To denature the template, 90-95°C must be reached for a short interval.
Prolonged incubation times at this temperature will lead to severe enzyme
losses by degradation. It was demonstrated (43) that complete strand sepa-
ration is not a necessary condition for annealing, rather, a partial melting
of the strands is sufficient to permit hybridization of the primers. - The annealing temperature must be attained for a few seconds only.
At a primer concentration of 1 /aM, the half-life time of the template-
primer hybrid is not more than 0.4 s at a temperature 20°C below the
T m value (44). - As mentioned earlier, the incorporation rate of the enzyme is 60-150
nucleotides/s at 70-75°C; thus, a few seconds are adequate for the
amplification of sequences shorter than 1000 nucleotides. An incuba-
tion time of 1 min/1000 nucleotides of sequence length is a good rule of
thumb, when sequences exceeding a length of 1000 nucleotides or more
are to be amplified (24).
3.2.6. Standard PCR Protocol
Following this protocol, a product band should be detectable from
a single copy gene. Prepare the following solution in a final volume of
30-100 lxl_,:
- I lag Genomic DNA as template;
- 1 ~tM Primers, 20-25 bp in length, flanking a sequence of 100-1000 bp;
- 10 mM Tris-HCl, pH 8.4;
- 50 mM KCI;
- 1.5 mMMgCI2;
- 0.01% Gelatine; and
- 200 pM of each dNTP.