Dauer Pheromone Prep

Authors: From Cori Bargmann and Brenda Reinhart 1992
A variation of Riddle's protocol:

Small molecule prep-
1. grow up worms super dense

1 liter of culture of worms in s-basal and e.coli
grow for 7-10 days, letting it clear once, feed it more e.coli and let it clear again

2. spin worms out

3. filter supe through whatman paper and then through sterile filter

4. boil supe down in a 2 liter flask to about 50 ml

5. spin put insoluble junk, filter and boil to 10 ml

6. pin put junk and boil to dryness-yellowish crust

7. extract it with 10ml ethanol overnight after breaking it up with a spatula into little yellow crusts

8. repeat with several O/N extractions

9. speed vac the etoh to dryness

10. resuspend in 10ml water and freeze

Note: when wash worms, be very careful to avoid contamination and wash E.coli in s-basal once before you add them

Cleaner prep: but lower yield
1. grow up a mess a worms from 1 liter culture

2. spin out worms and keep them

3. float them through sucrose to get rid of junk

4. put worms in 250ml flask with 50ml s-basal and leave at 20 degrees in shaker o/n

5. spin out worms

6. filter supe and freeze