Sucrose Gradient for Worm Separation

Model Organism: Worms Methods & Reagents
1

Hi everyone,

I’d like to use a sucrose gradient to separate different worm stages. Before I start testing different sucrose layers, I’m hoping someone here has already figured out the best %'s to layer for separation. Any help would be greatly appreciated

Chris

2

Hi,

save on sucrose (sugar is bad for you) and instead read the WBG article on filtration;

http://www.wormbook.org/wli/wbg4.1p16/

Regards

Steve

3

Thanks. L3’s appear fairly terrible to separate by that method though. Would it be best to isolate L1’s and time their growth then? I’ve been doing that by taking an egg prep, halting them at L1
via starvation, then transferring them to a liquid culture for x hours until the desired stage is reached. I’m going to be running the different stages through a mass spec so having nearly pure populations is crucial.

4

Hi,

Synch’ing worms comes up regularly, so if you want to go down the ‘synch, grow & show’ route then take a look at this excellent summary posted on the forum a while ago.

http://forums.wormbase.org/index.php?topic=344.msg527#msg527

I guess the main point for your experiments is that you will always have some spread in ages, but picking staged adults and moving them as described reduces this variation such that you can obtain pretty pure, staged populations.

Using the synch’ing approach will give good results and isn’t as messy or lossy as attempting the same using a sucrose gradient or the nylon filter method. So yes, try the synch’ing approach.

Steve