Two long PCR steps:
-
First round of the nested PCR step of the end point dilution procedure to quantitate the cDNA.
-
First round of the nested PCR step of the limiting dilution step to amplify single templates. The conditions are exactly the same both times.
Reaction Composition
| H2O |
35.5 µL |
|
| 10x Buffer + 15 mM MgCl2 |
5.0 µL |
final [Mg2+] = 1.5 mM |
| dNTP |
6.0 µL |
final [dNTP] = 300 µM each |
| DS3 (4895-4924) |
1.0 µL |
final [primer] = 0.2 µM |
| DS8 (9550-9521) |
1.0 µL |
final [primer] = 0.2 µM |
| Enzyme (3.5 U/µL) |
0.5 µL |
final enzyme amount = 1.75 U |
| Template (cDNA) |
1.0 µL |
|
|
|
|
| Total |
50.0 µL |
|
Primer positions are given for NL4-3, and yield a product ~4.6 kb. The enzyme and buffer are from Boehringer Mannheim?s Expand? High Fidelity PCR System.
Cycling Conditions
-
94°C for 2 min, 30 sec
-
94°C for 15 sec - 55°C for 45 sec - 68°C for 6 min for 9 cycles
-
94°C for 15 sec - 57°C for 45 45 sec - 68°C for 6 min, 20 sec for 19 cycles
-
72°C for 30 min
-
4°C soak
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