192
VOLKER JUNG and GEORG A. WEBER
Standard PCR amplification protocol using Taq polymerase
1. Add to sampled and digested cells:
- 2.5 J..lI Taq-buffer
- 2.5 J..lI dNTP-mix (2 J..lM)
- 2.5 J..lI DOP-primer (10 J..lM)
- 0.5 J..lI Taq polymerase (2.5 units)
2. Fill with ultrapure water to a volume of 25 J..ll.
3. Proceed in a thermocyder according to the following PCR program:
94°e
4 min
94°e
1 min
30
0 e
1.5 min
Up to n °e
within 4 min
noe
3 min
94°e
1 min
56°e
1 min
noe
3 min
noe
5 min
20
0 e
endless
(8 cycles)
(35 cycles)
Two-step PCR amplification protocol using ThermoSequenase and Taq polymerase
1. Add to sampled and digested cells:
- 1 J..lI ThermoSequenase reaction buffer
- 1 J..lI dNTP-mix (2 J..lM)
- 1 J..lI DOP-primer (10 J..lM)
- 1 J..lI ThermoSequenase (4 units)
2. Proceed in a thermocyder according to the following PCR program:
94°e
4 min
94°e
1 min
30
0 e
1 min
Up to noe
within 4 min
noe
3 min
(8 cycles)
4°e
endless