External Round | Internal Round | ||
|---|---|---|---|
| Master Mix: | Master Mix: | ||
| 1 µl | dNTP mix (2mM each dNTP) | 1 µl | dNTP mix (2mM each dNTP) |
| 1 µl | 10x PCR buffer (15mM MgCl2) | 1 µl | 10x PCR buffer (15mM MgCl2) |
| 0.04 µl | Taq polymerase (0.2U) | 0.04 µl | Taq polymerase (0.2U) |
| 5.16 µl | dH2O | 7.16 µl | dH2O |
| 0.8 µl | primer mix (5 pmoles/µl each, forward & reverse primer) | 0.8 µl | primer mix (5 pmoles/µl each, forward & reverse primer) |
| DNA: | DNA: | ||
| 2 µl | containing DNA (typically sample of worms or single worm digested in lysis buffer followed by heat inactivation of Proteinase K) | 0.2 µl | sample from external round |
| Cycling Parameters (external and internal PCR) |
|
|---|---|
| 1 cycle: | 94ºC/30 sec. |
| 35 cycles: | 94ºC/30 sec. 55ºC/30 sec. 72ºC/30 sec. to 120 sec.∗ |
| Hold: | 4ºC |
∗ We use parameters that let us see the wild-type product. 30-second extension time generally is adequate for products up to 1.2 kbp. Use 60 seconds for products up to about 2.4 kbp, and 90 or 120 seconds for larger products. In some cases these times will prove unsatisfactory, and you will want to adjust them up or down.