Hi all,
I have the strain, sod-3::gfp which I kept frozen @ -80 for last few months.
Now when I thaw it, the worms are hardly growing and even the adults are dying after few days without hatching of eggs…
I have couple of more vials, which I will thaw again… So any suggestions of how to keep them growing normally ?
Thanks.
The younger larvae survive freeze/thaw better than older animals. It sounds like when you froze them down, the vast majority of animals on the plate(s) were older. I would thaw all the vials to try to recover any really young animals that may be in there. If you do get them back, when you re-freeze the stock make sure the plate has a lot of younger larvae.
There’s really nothing tricky to thawing elegans (though if you’re thawing the tubes in a water bath, say at 37 degrees, one possibility is that you’re leaving them in there too long; I’d recommend thawing tubes in your hand or putting them in a 37 degree water bath just until the outer part of the chunk of frozen worms thaws - perhaps 30 seconds, and certainly not more than 60), so as Morris says the most likely explanation is that the initial freeze wasn’t good, possibly that it wasn’t mostly a population of starved L1s. I would add that when you have frozen a strain and are transferring it to its long-term storage you should do a test thaw (either of an extra vial or of a chip from the vial if you’re using the soft agar method) to make sure the frozen worms are good, while you still have the strain growing in case they’re not. If the test thaw is good, the worms left at -80 should be good for a long, long time.
Given that a couple of attempts to thaw this strain have failed, you might want to test thaw other strains you’ve frozen.
I’m with Hillel. I test thaw every single strain I freeze, usually 2-8 days later. Sometimes bacterial contamination during freezing will make for a bad freeze. Or bad freeze solution. Thaw onto large plates so glycerol concentration is lower–worms don’t like glycerol. Freezing while there is still food on the plate (and thus L1s don’t enter L1 diapause) is a problem. Likewise, daf-16(lf) strains freeze very poorly if you starve them more than a day, because they fail to arrest cell divisions in diapause. We even had a certain promoter::activated transgene combo that froze poorly: transgenics only, as their non-transgenic sibs recovered nicely.
So, always check the freeze while you still have a parafilmed strain. You never know! We would have lost that transgene had we not test thawed.
David’s point about daf-16(lf) worms may be especially relevant, as your sod-3::gfp transgene may be soaking up DAF-16.
Great point, Hillel! That seems like a plausible trouble spot, particularly if the non-array-bearing animals recovered well.
Thanks a lot everyone for your excellent replies…
Really greatful…