swPCR of F1 SAS-6

Searching for F1 rollers and small worms

On my final days in the Griffin lab, worms were injected with the SAS-6 injection mix that I had prepared. The F1 worms from the injected worm was individually picked and plated. I was able to practice this by identifying rollers and worms smaller than rollers. This was partly difficult as although the roller phenotype is typically very obvious (indicated by the circular trajectory of the worm movements), some rollers had a less obvious phenotype. To identify these rollers I had to observe if the worms lifted their heads off the plate - this would indicate a roller. I then picked all the rollers and smaller worms, allowing them to grow on their own individual plate and to have their own progeny. After a few days, I then picked the grown F1s and placed them into 20ul of 1x PCR buffer, repeating the same steps as my previous swPCRs.

conducting swPCR on 70 samples of SAS-6 F1s

These worms were lysed, amplified and then PCR products were ran on a gel. As there were 70 samples of worms to process, this protocol was quite long, however with my previous experience with SPD-2, and helping Dr Sally Adams with swPCR for MOM-2, I started getting a bit more used to pipetting for a longer time.

After swPCR and running the gel I was able to identify heterozygotes for SAS-6! Initially, out of the 67 worms screened, 5 displayed the 955bp band we expected to see. From those 5 I reran the gel to confirm whether or not the insert was present. This secondary gel demonstrated 3 worms with the insert, worm 8, 14 and 20. Despite worms 24 and 38 displaying a clear second band, the band was not near the expected band size, and therefore we stuck with the 3 worms. 8 F2 worms from the selected worms were picked and allowed to grow, which would then be rescreened in hopes to identify a homozygote strain.

heterozygote worms with SAS-6 insert at the expected band size (955bp), found in worms 8, 14 and 20.