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PCR
Procedure and Conditions for the PCR
We have created this table to provide newcomers
to the PCR with an overview of the common rules and concentrations which
should be observed when designing new experimental set-ups.
There are, of course, a number of exceptions
and special points which, due to lack of space, cannot be covered here.
Feel free to send us any general suggestions or tips on this subject,
they are greatly appreciated.
| Steps |
Basic rules |
Comments |
1
No. of cycles |
| No. of source molecules |
No. of cycles |
| 105 |
25–30 |
| 104 |
30–35 |
| 103 |
35–40 |
| 50 and less |
20–30, followed by a
second PCR with "nested" primers, i.e. with
a primer pair which binds between the first two primers
of the target sequence |
|
To prevent the formation of nonspecific
products, do not carry out more than 40 cycles |
2
Denaturation step |
- 95°C for 30 sec. or 97°C for 15
sec.
- For complex templates (genomic DNA) begin
with denaturation for 5–10 min. before the actual cycles
(Hot Start)
- G/C content greater than 50% increases the
denaturation temperature
- T1/2 = half life of Taq at specific temperature
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Inefficient denaturation
is a frequent cause of errors. However:
- T1/2 Taq at 92.5°C = 2 hours
- T1/2 Taq at 95.0°C = 40 min.
- T1/2 Taq at 97.5°C = 5 min.
- T1/2 Taq at 97.5°C + 10% glycerin =
23 min.
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3
Annealing step |
For standard primers
(approx. 20 nucleotides [nt]; 1 µM; 100% match)
- Approx. 20 sec.
- Tm = melting point
of DNA
- Ta = annealing
temperature
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- Low concentrations and long primers extend
necessary annealing times. The annealing temperature (Ta)
can be estimated on the basis of the melting temperature
(Tm) using the following formula:
- Tm = (A+T) x 2
+ (C+G) x 4 [Wallace], up to ca. 20 nt
- Tm = 81.5°C
+ 16.6 (log[Na+]) + 0.41 (%G+C) – (500/n) – 0.61
(%FA) [Meinkoth and Wahl]; FA = Formamide
- Ta = Tm
– (5 to 10°C)
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4
Elongation step |
- Approx. 60 bp are synthesized every second
under optimum conditions
- 2 Kbp require approx. 1 min.
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- The shorter the fragment, the easier it
is to control the reaction
- 200–500 bp fragments are sufficient
for most detection reactions
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