| External Round
Master Mix:
2.5 ul dNTP mix (2mM each dNTP)
2.5 ul 10x PCR buffer (15mM MgCl2)
0.1 ul Taq polymerase (0.5U)
11.9 ul dH2O
2 ul primer mix (5 pmoles/ul each, forward & reverse primer)
1 ul poison primer (10 pmoles/ul)
|
Internal Round
Master Mix:
2.5 ul dNTP mix (2mM each dNTP)
2.5 ul 10x PCR buffer (15mM MgCl2)
0.1 ul Taq polymerase (0.5U)
17.4 ul dH2O
2 ul primer mix (5 pmoles/ul each, forward & reverse primer)
|
DNA:
5 ul containing DNA (typically sample of worms or single worm digested in lysis buffer followed by heat inactivation of Proteinase K) |
DNA:
0.5 ul sample from external round |
Cycling Parameters
(external round)
|
| 1 cycle: |
94°/30s |
| 35 cycles: |
94°/30s
61°/30s
72°/30s to 120s* |
| Hold: |
4° |
|
Cycling Parameters
(internal round)
|
| 1 cycle: |
94°/30s |
| 35 cycles: |
94°/30s
55°/30s
72°/30s to 120s* |
| Hold: |
4° |
|
