374
JINGLY FUNG et al.
2. Denature 4 III DNA (template) at noc for 6 min.
3. Add 8111 T7 Mix. Place at room temperature for 2 min, and then at 37°C
for 6 min.
4. Denature at noc for 3 min.
5. Add 2111 T7 Mix. Place at room temperature for 2 min, and then at 37°C
for 6 min.
6. Repeat steps (4)-(5) 5 times in total.
7. Denature DNA at noc for 3 min.
8. Add 8111 T7 Mix. Place at room temperature for 2 min, and then at 37°C
for 10 min.
9. The products at this stage can be used immediately for the Taq polymerase amplification reaction or stored at -20°C for up to several years.
10. Taq polymerase (35 cycles) PCR:
In a 0.5-ml tubes mix in this order (for a total of 200 Ill)
- 152 III water
- 20 III 10 x Taq buffer
- 4 III 4dNTPs
- 4 III primer JUN15
- 20 III Sequenase reaction product
- 0.67 III Taq
11. Run 35 cycles of the following PCR reaction (Perkin Elmer thermocycle
file).
Temperature (DC)
94
94
52
52
72
72
72
Time (s)
30
60
60
60
120
120 (plus 5-s extension per cycle)
600