Hello,
I have to set up a bunch of RNAi lifespans on different HT115 clones and would like to ask how others usually rear the worms?
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Egg lay or bleach worms onto OP50 till L4, then transfer to RNAi plates with induced HT115 for each gene of interest cognizant of the fact that there will be some bacterial strain (OP50 + HT115) mixing?
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Egg lay or bleach worms onto HT115/L4440 and at L4 transfer to HT115 clone of gene of interest.
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Some other way?
I transfer at the L4 stage to avoid developmental issues of k/d before this stage. I have also tried rearing worms for 3 generations on L4440 (rather than OP50), then egg laying into L4440 and transferring to HT115 gene of interest RNAi at L4. I find that the worms develop differently (grow slower when I do this vs growing on OP50). I am using strain NL2099, which has been backcrossed twice. One suggestion I got is to transfer the worms onto an unseeded plate from an OP50 seeded plate for 20-30 mins right before transferring to RNAi plates. I am really not a fan of this idea for two reasons: 1) I think that would still transfer some OP50 and 2) it would be very inconvenient to do given the nature of the project. How do others who do RNAi experiments deal with this? Thanks!