C. elegans Lysis PCR

Materials

  • Lysis solution
  • Thermocycler
  • Minicentrifuge
  • 20 mg/mL proteinase K

Protocol

  1. Prepare lysis solution by combining 98 μl of 2X Lysis Buffer (recipe below) with 2 μl of 20 mg/mL Proteinase K (PK).

    a. Prepare enough lysis solution for the number of lyses you want to carry out (6 μl per reaction).

  2. Aliquot 6 μl of the lysis solution into each cap of an 8-tube strip cap.

  3. Pick desired number of worms into each cap (3-5 worms per cap).

  4. Put the caps onto an appropriately labeled strip of tubes. Spin down the worms into the 8-strip tube using the minicentrifuge.

  5. Run the following program on a thermocycler:

Time Temperature Cycles
1 hour 60°C 1
15 min 95°C 1
Hold 6°C 1
  1. When there is about 20 minutes left on the lysis, set up your PCR master mix (number of reactions + 1):
Constituent Stock Volume Final
Lysis reaction - 2 -
Forward primer 10 μM 2 0.1 μM
Reverse primer 10 μM 2 0.1 μM
dNTPs 2.5 mM 1.6 0.2 mM
5X Buffer 5X 4 1X
Taq - 0.2 -
H2O - 8.2 -
  1. Add 18 μL of master mix to each tube of a strip of tubes.

  2. When the lysis is done, add 2 μL of the lysis to each reaction. Keep reactions on ice until the thermocycler is ready.

  3. Run the following program on a thermocycler:

Time Temperature Cycles
3 min 95°C 1
15 sec 95°C -
15 sec Primer Tm - 5 35
1 min 72°C -
5 min 72°C 1
Hold 6°C

Recipe for 2X lysis buffer

Constituent Amount Final
KCl 0.745 g 100 mM
Tris (pH 8.2) 0.24 g 20 mM
MgCl2 0.047 g 5 mM
IGEPAL 900 μL 0.9%
Tween 20 900 μL 0.9%
Gelatin 20 mg 0.02%
  • Add water to 100 mL

  • Note: Tween 20 and IGEPAL are difficult to pipette, so you may choose to make a 50% solution in water and double the input amount.