Pcr

1. Set up the following reaction (30 |L) by adding the reagents in this order: 500 ng of a genomic male patient DNA, 500 ng of a male control DNA (see Note 5), H2O, dNTP's mix (25 mM), 12.5 pmol/|L of each oligonucleotide primer (Table 2, see Note 6), 10X Super Taq buffer, 15 mM MgCl2 (see Note 7) and 1.5 U AmpliTaq DNA polymerase (5 U/|L).

2. PCR is performed in a thermal cycler (DNA Engine Tetrad, MJ Research) using the following conditions (see Note 8):

Initial denaturation 94°C, 5 min Stage I (5 cycles) 94°C, 30 s denaturation

52°C, 30 s annealing 72°C, 1 min extension

Stage II (5 cycles) 94°C, 30 s denaturation

48°C, 30 s annealing 72°C, 1 min extension

Stage III (25 cycles) 94°C, 30 s denaturation 45°C, 30 s annealing 72°C, 1 min extension

Stage IV (Final) 72°C, 5 min extension

94°C, 10 min denaturation 50°C, 1 min annealing 4°C, soak

Was this article helpful?

0 0

Post a comment