Standard Nested PCR Protocol       

25 ul reactions, amounts per reaction Make MM for 1 extra reaction every 6

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) 12.9 ul dH2O 2 ul primer mix (5 pmoles/ul each, forward & reverse primer)

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 and internal PCR)

1 cycle: 94°/30s

35 cycles: 94°/30s 55°/30s 72°/30s to 120s*

Hold: 4°

*We use parameters that let us see the wild-type product.  30-second extension time generally is adequate for products up to 1.2 kb.  Use 60 seconds for products up to about 2.4 kb, 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.