Help with c.elegans egg isolation - (Nov/05/2004 )
protocol needed to isolate eggs from a mixed population. bleaching was tried but was unsuccessful. worms died but were not digested. suggestions would be greatly appreciated.
I use a mix 20ml clorox, 80 ml dH2O and 1g NaOH. Dissolves worms but may be to strong b/c smaller yields.
i was doing in egg preparation according to the following protocol with great sucess:
Flush plate with worms on in with 4 ml steril ddH2O
pipet solution in 3 2ml tubes
add 500 µl NaOCl solution
vortex every 2 min for 10 min
centrifuge 30 s at 3200 rpm
pipet supernatant off, leave 100 µl
add 1 ml sterile ddH2O
vortex again, centrifuge again
resuspend pellet in a few drops M9 buffer
ddH2O 0,6 ml
NaOCl (12 % cl) 0,5 ml
6M NaOH 0,4 ml
1 M MgSO4 1 ml
ddH2O ad 1 litre
Hope that helps
I would bleach for longer. Watch the worms under your scope. You will see them break open. If that does not work, get as many as you can, cut them on glass with a razor and strain this prep through a 50 micros filter, collect on a less than 15 micron filter and you should get all eggs.
Sometimes you can't bleach because of downstream applications. Here's what I do:
get a clean piece of glass and ethanol it. Rinse with diH20.
Pipette DDiH20 onto the glass ~300 ul. Pick gravid adults into the water. take a razor and while looking under your scope, physically cut the worms, just one cut per worm should do, the ddwater will make them pop open. Then using a mouth pipette tube and a drawn capillary tube pick out individual embryos. It does take some skill and your viability will be less than optimal at first, but you can get 100% with some practice. The most important thing is sizing the needle so as not to get too much water but not crush the egg.
I have heard you can even pick what stage you want your embryos this way, but that is beyond me.