Long PCR
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Two long PCR steps:
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First round of the nested PCR step of the end point dilution procedure to quantitate the cDNA.
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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
| H2 O | 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 | |
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| 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
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94°C for 2 min, 30 sec
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94°C for 15 sec - 55°C for 45 sec - 68°C for 6 min for 9 cycles
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94°C for 15 sec - 57°C for 45 45 sec - 68°C for 6 min, 20 sec for 19 cycles
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72°C for 30 min
4°C soak
Sensitivity
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Presumably one copy of pNL4-3 and one copy of cDNA.
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Provirus (8E5 cells, with one provirus per cell) down to 100 copies.
Specificity
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Nothing detected from uninfected human placental DNA.
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No apparent false priming
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PCR/RT-PCR/qPCR
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AFLP分析
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