Schedl Lab

Washington University Genetics

Worm Powder

Modified by Sudhir Nayak from unknown original

1. Put 15 – 20 adult hermaphrodites on two 10 cm NGM plates and starve until L1s accumulate.  Transfer each to 100ml of liquid culture as usual and incubate 3-4 days at 20deg with shaking.

2. Harvest solutions; cold 0.1 N NaCl, cold ddH2O, cold 60% sucrose

a) Spin down worms; GSA rotor, 2.5 K rpm, 2 min (cold)

b) Collect into 50 ml falcon tube, spin down 1K, 1min and wash 2 times with cold 0.1 N NaCl.

c) Sucrose float worms as usual

d) Collect floating worms into cold water (~15 ml), spin down and wash 2x with cold water

3. Fix with 10 ml of 3% formaldehyde / 0.1 M K2HPO4 (pH7.2) (Prepare from sealed ampoules of 16% EM grade formaldehyde (EM Sciences). Freeze any excess.) for 4 hrs.

4. Spin down and remove formaldehyde, then wash with PBT (PBS+0.1% tween) 2x.

5. Post-Fix with cold (-20) Acetone for 20 min, then wash with PBT 2x times and with PBS 2x.

6. Bring up the volume to 10 ml with PBS, then French press twice at 12, 000 PSI to make worm powder.

7. Wash 1x with PBS and dry under vacuum (Lyophilization is better).

8. Starting point to absorption.

Dilute serum with PBT (1:1, 500 ul serum and 500 ul PBT) and add about 100 ul worm powder (It should become a dense slurry).

Rotate O/N at 4 to absorb cross-reacting antibodies.

Spin down 14,000 rpm for 10 min at 4.

Collect supernatant and test it!!

  • Tim Schedl, Ph.D
    Department of Genetics
    Campus Box #8232
    Washington University School of Medicine
    4566 Scott Ave.
    St. Louis, MO 63110

    Contact

    Email: ts@genetics.wustl.edu
    PHONE: (314)362-6164 [lab]
    FAX: (314)362-7855