So yesterday, while looking at my fruiting bodies, I found out that almost all of my plates from one transformation exhibited a phenotype similar to P13k1/2- mutants. I remember plating my HO12 PTEN- mutants on Ka labeled P13K1/2 because my flask of Ka labeled PTEN disappeared…and that shouldn’t have done anything unless that flask of Ka was contaminated with P13K1/2- mutants. However, even if it was contaminated, all of those mutants would have died in the cold room where the flasks of Ka are stored. What’s more likely is that I transformed them into P13K1/2- mutants instead of PTEN. Perhaps I thawed out the wrong cells when I plated them for transformations initially, perhaps the HL5/hygromycin media I used was contaminated with P13K1/2- mutants, perhaps I used the wrong plate…
But! What matters now is that we discovered this, and chucked all of those platand their respective data from fruiting body assays or microscopy away. It’s like they’ve never existed…all those long hours gone. Am i going to get some of that sleep back? Anyways, one batch of cells exhibiting this phenotype on S&M plates should be fine, because they were transformed over 3 weeks back. Therefore maybe the contamination happened later. I replated them on plates with fresh Ka today, and I will be able to see the results Monday.
Other than that, I have been in the microscopy room almost everyday. I’ve lost count of how many videos I’ve done. I know I recorded 5 movies today after the meeting, and incubated 11 different strains of PTEN- mutants for microscopy tomorrow morning. I will take a lunch break, then record another 3 in the afternoon. Afterwards, I’ll incubate another 11 PTEN- strains for microscopy Friday morning, and record 12 WT strains in the afternoon. I also need to split my cells before the weekend. I only have about 60 now thanks to the P13K1/2- contamination.
I think I would rather have more cells, even though it’s more work, because then I would have more data. Then again, I feel like I’m swimming in my data. I have so much, and still so much more to collect that I haven’t even had time to really sit down and analyze them! And that’s the most exciting part too.
If I analyze my data after I finish everything, I think I will be done around 9:30 and I don’t want to be stranded at UCSF at night…although, that IKEA couch might be more handy than I realized!
On top of my workload, I also have iGEM homework. I should get started on my glossary.