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.
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.
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…