I received a gene deletion strain from CGC a few days ago and immediately did single-worm PCRs, diagnostic for the deletion, using 10 adults taken directly from their plate. Assuming I did everything correctly – and I’m sure I did – one of these ten gave a single PCR product consistent with wild-type (N2) and a second gave two products suggestive of heterozygosity. The remaining eight gave a single product possible only with the deletion. I’m going to repeat these PCRs but I guess I should single some worms and generate pure lines homozygous for the deletion – yes?
It would be helpful to know the strain you received, so we can look up the genotype to help troubleshoot your problem. But from what you said, yes, that’s what you should do.
I imagine if you got homozygous bands from picking larvae/adults this deletion is expected to be homozygous viable, but if it isn’t then you would have received a balanced het from the CGC, in which case you may expect the profile you got.
Steve
I would say that it seems your strain isn’t homozygous as the CGC description suggests. Are the WT bands you see as intense as the deletion bands (ie, are you sure they are real products?) I would suggest doing as you suggested, cloning out and doing single worm PCR to genotype and then check the next generation for proper segregation.
Yes that’s listed as homozygous viable. It’s odd that you didn’t receive it in that condition, but you’ll want to homozygose it in the way you suggested. Then you should outcross it to remove background mutations.
Good luck!
Steve
Colton,
And please do notify the CGC about your problem. Problems occur very rarely, but when they do it is VERY important that the CGC knows that there is a problem - they will work immediately to fix it.
Janet
After rereading the original post, an alternative possibility comes to mind. The ratios of observed genotypes are fairly skewed for a heterozygous strain - the majority should produce both PCR products. It’s possible that the strain was contaminated with a second one, but then you’d expect to see only homozygotes (unless the contaminant contained males, but then you’d expect to see males on the plate). Not to impugn Colton’s technique, but contamination of the PCR with WT DNA might better explain the data.
-Harold