Long PCR

Two long PCR steps:

1. First round of the nested PCR step of the end point dilution procedure to quantitate the cDNA.

2. 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

1. 94°C for 2 min, 30 sec

2. 94°C for 15 sec - 55°C for 45 sec - 68°C for 6 min for 9 cycles

3. 94°C for 15 sec - 57°C for 45 45 sec - 68°C for 6 min, 20 sec for 19 cycles

4. 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