Adapted by Sudhir Nayak from various originals.
This whole procedure is performed in PCR tubes (200ul), strips, or plates. There are no spins and the proteinase K digestion is heat inactivated so the prep is ready for PCR with no additional clean up necessary. Most robust and reproducible results are obtained in single worm PCR when using nested primer sets.
Procedure:
1) Make 100ug/ml proteinase K solution in WLB.
2) Place 5 ul of lysis buffer in the bottom of a PCR tube. As little as 1ul can be used but its harder to get the worm off the pick.
3) Pick single worm (or a few worms) directly into the tube with lysis buffer + 100ug/ml proteinase K. As many as 20 worms (or more?) can be used with this prep method.
4) Worms can be frozen @ -80 indefinitely. The freeze/thaw may help lysis but is not absolutely necessary.
5) Heat tube to 60deg for 60min.
6) Inactivate proteinase K by heating to 95 degrees for 15min.
7) Add your PCR mix directly to the digested heat inactivated prep and cycle as usual (30-35x).
8) Add 2.5ul of DNA blue juice to PCR reaction and run 10ul on gel for analysis.
Worm Lysis Buffer (WLB)
50 mM KCl
10 mM Tris pH 8.3
2.5 mM MgCl2
0.45% NP-40 (IGEPAL)
0.45% Tween-20
0.01% Gelatin
Proteinase K stock solution
20mg/ml proteinase K