Ron Lab


 

PCR Analysis of Dead Eggs from C. elegans

Cristina Benedetti, Skirball Institute

  • The presumptive dead eggs are transferred with a platinum wire to a new seeded plate for an overnight incubation.
  • The day after, the unhatched eggs (defined as dead) are transferred to a new unseeded plate and cleaned as much as possible from the bacteria .
  • A glass capillary tube is filled with 5 µL of worm lysis buffer. Then, exploiting capillary action, the dead egg is picked up. The contents of the capillary are expelled into an eppendorf tube containing 3 µL of lysis buffer (by gently blowing on the opposite end).
  • The eggs in the lysis buffer are placed at –80 deg C for at least 5 minutes (they can be kept frozen for days): this step helps crack the eggs.
  • The eggs lysate is incubated at 50 deg C for an hour, at 95 deg C for 15 minutes and then centrifuged at max speed for 1 minute.

Buffers

Worm lysis buffer: 50 mM KCL, 10 mM Tris-HCL pH=8.3, 2.5 mM MgCl2, 0.45% NP-40, 0.45% Tween 20, 0.01% gelatin, freshly added 60 mg/ml proteinase K).

PCR conditions (total volume: 10µL)

1 µL eggs DNA

1 µL PCR buffer

1 µL dNTPs 2.5mM

0.33 µL primers (5mM)

0.05 µL Taq polymerase

6.29 µL H2O