After synchronization, there are still some worms in different periods. Does anyone know why? My worms is a N2 strain.Thanks!
Can you say more about the protocol you’re using, and the conditions? There are a bunch of variants.
My own preferred protocol - I’m not entirely sure of its provenance - is as follows:
1 ) wash gravid worms and eggs off a plate or plates in water into a 15 ml conical tube. Pellet them, 1 minute at 1k rcf.
2 ) Aspirate supernatant, rinse once or twice in water, spinning down and removing supernatant as before
3 ) Add a couple of ml from the following freshly made solution: 4.05 ml H2O, 850 ul 5M NaOH, 250 ul sodium hypochlorite solution
4 ) Suspend the worms and occasionally resuspend them by periodic shaking or swirling, but not excessive vortexing
5 ) Once there are no worms visible to the eye, just bright specks (typically 8-14 minutes depending on density and species) spin down the eggs as before (1 minute, 1k rcf)
6 ) Aspirate supernatant, Add 5 ml M9, spin down
7 ) Repeat previous step for a second M9 rinse
8 ) Add 0.5-1 ml S medium. Place tube(s) on a wheel at 20C to hatch overnight.
9 ) Start aliquots growing on seeded plates.
If properly done, bleaching for eggs and letting them hatch in the absence of food and enter L1 arrest should give you quite a tightly synchronized population, at least for a healthy, consistent strain. If you start arrested wild-type worms growing on food, the vast preponderance of them should reach adulthood within an hour or two of each other.
Castro et al points out that the ethanol used to add cholesterol to S medium can act as a carbon source, which may not be desirable for the very tightest possible starvation, but this is unlikely to be an issue for most applications.
Thank you for your kind reply！ Methods in my experiment：
About 20`30 adult hermaphrodites were transferred onto fresh NGM plates, allowed to lay eggs for 2~3 h and then removed. Wroms was cultured in 20 ℃.
In addition,my ht1593 have the same phenomenon. About half of the ht 1593 worms are at other times after synchronizationThe method you describe has the virtue of not starving and arresting your L1s, but it’s less reliable and there are important caveats and details.
If you’re going to roughly synchronize one strain that way, you need to make sure the adults are at the same age: younger adults will lay younger (more recently fertilized) eggs than will older adults. You’ll want to pick late L4 animals the night before you use them as egg laying adults.
It gets more complicated once there’s more than one strain: how sure are you that the two strains are similarly fertile, and are laying eggs of the same developmental stage? Might a mutant strain in particular have some variability from animal to animal for egg laying and fertility?
You can help things a bit by at least checking that the eggs are roughly synchronous by looking at their morphology, or you can roughly synchronize by hand-picking eggs based on their moprhology - hand-picking comma-stage embryos, for example.
Thanks， I’ll consider your proposal carefully :). Do you know whether passage number have an effect on synchronization?
- We usually put 20 ~30 gravid adults on NGM plates and let worms lay eggs at 25C for 45 minutes. (2 or 3 hrs are too long. if you need more eggs, you can put more gravid adults on your plates.
- Remove gravid adults.
- let eggs hatch and grow at 20C.