Hi everyone,
I’m PhD student working with C. elegans from 1 year. I have some problems with synchronization the culture.
We’ve decided to change a litlle bit the methods and i’ve repeated it few times and still there is something gowing wrong.
At the begining, sory for my english but I hope I’ll be well understood:)
I read all the post about synchronization, trying all the metods and what I’ve got:
After keeping eggs in M9 on the wheel for a night, I can’t see any of L1 stage. I think the problem is that eggs are in big amount of liquid.
Now I’m trying to keep on the plate without bacteria and on the 12-well plate, and after night, or 24 hours, I still can see some eggs or I’ve got another porblem.
On the plate there are few L1 and when I’m trying to trasfer them to the plate with OP50 I have large amount of liguid or almost empty plate. Is there any method
to have a huge number of nematode? Can I spin them? I need to have a lot of worms in small value of liquid.
Again, If, luckily, I have few nematode, after 48 h on the plate with ngm and OP50 most of them are dead.
What am I doing wrong?
Can somebody please halp me?
I would be very appreciate.
Thanks
Marta
I’m using 0,5 ml 5 M NaOH, 1 ml NaOCl for each 3,5 of supernatant and bleach it for 7 min.
How U remove supernatant? Can I spin it? Or is there eny other method to allow nematodes to settle at the bottom?
I still have very small amount of L1.
Thanks a lot
After bleaching the worms for 5 minutes, you can spin down the eggs at 1.5 g for 1 minute and then decant the supernatant. Follow this with 2 washes in M9, and then bring the eggs up in your final volume in M9 (I usually do ~10-12 mL in a 15 mL conical). Gently rock the sample @ RT, and 16-24 hours later pellet the L1s at 2k rpm for 2 minutes. The L1s will pellet best using a clinical centrifuge with the brake off. Carefully remove the supernatant, leaving ~100 ul M9. This should leave you with a well concentrated batch of synchronized L1s.
the magic of the worm community forum is the SEARCH function…you type in synchronization (or its English equivalent) and you get about 10 hits…
Discussions about synchronisation (that’s better) have been going since I was a boy (well almost). So it is always best first to check what ‘wise’ people have already said;
Don’t change methods that work until you have them working!
Only change the methods when there is an advantage in doing that.
Use fresh solutions, chuck old ones out and stick to the timing. If it says vortex each minute then do it, if it says bleach until the worms disappear…then that’s what you do.
Look through the worm methods book and you will see lots of methods that have been used over the years and work.
Thank You all for advises:) I will keep going and I hope one day it will be fine.
Dear steveh, believe me, i would’nt ask if it didn’t take so much time…
but thanks:) for good advices.
i would check everything
THANK YOU GUYS, ONCE AGAIN:)
hey, no problem…just pointing to the search function as a useful starting point in future visits. We have all been there where a method that has been working for a long time stops working. Being methodical and working through the problem takes a lot of time I know…
