Long PCR
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 sec - 68°C for 6 min, 20 sec for 19 cycles
72°C for 30 min
4°C soak
Sensitivity
Presumably one copy of pNL4-3 and one copy of cDNA.
Provirus (8E5 cells, with one provirus per cell) down to 100 copies.
Specificity
Nothing detected from uninfected human placental DNA.
No apparent false priming