(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 .

1-2 dead eggs are picked up with a borosilicate capillary tube (WPI, item # 1B100F-4) filled with 0.5ml of worm lysis buffer (pick-up is by capillary action) and transferred to an eppendorf tube containing 3ml of lysis buffer (the worms are expelled from the tube 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 minutr.

1ml of crude lysate supernatant is used for each PCR assay.

Buffers:

Worm lysis buffer: 50mM KCL, 10mM 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: 10l):

1ml eggs DNA

1ml PCR buffer

1 ml dNTPs 2.5mM

0.33 ml primers (5mM)

0,05 ml Taq polymerase

6.29 ml H₂O