Week 6

7/23
Right when I got back from camp, I went straight to work, eager to get going with my experiments. Bryan told me this was going to be a boring week, which I later found out to be true, as I spent most of my time sitting around waiting. Anyway, while I had been away Bryan had moved my Clone A shEGFP cells and my EGFP shEGFP cells to 10cm plates. The third group of cells, Clone A shβ4, had hardly grown (as expected) and had to be moved down into a 24-well plate. Also, I prepared bacteria samples 1.2 and 1.4 for a maxiprep, because we still needed DNA from sample 1. Today I only spent about 45 minutes working in the lab, but had to wait until 2:00 before I could put the bacteria into the incubator so that they wouldn’t overgrow and stop expressing the DNA that we hoped they contained.

7/24
I split all of my 293 samples (4 of them) down 1:20 so that they would be ready for transfection. I then spent the rest of the day performing a maxiprep on samples 1.2 and 1.4, which I hate doing because it involves working with solutions that smell terrible and get everything sticky. Also, it involves over an hour and a half of waiting time, for incubating and centrifuging, which made me glad I brought an ipod. Finally, to make this day officially the worst day yet, after the long process of the maxiprep, neither sample yielded any DNA, and though I was glad to be able to go home, I was upset because I would have to repeat the whole process in two days.

7/25
I plated 2 wells of a 6-well plate with clone A cells to get ready for viral infection, then I replaced the media and added puromycin for selection into the Clone A shβ4 sample. I then replaced the media in 3/4 of the 293 plates with FT media (no antibiotics) and waited until 2:30 this time before I placed the 1.2 and 1.4 cell samples into the incubator for the night.

7/26
I added 2 mL of shβ4 virus to one of the Clone A samples in the 6-well and 2 mL of media to the other sample as a control. I spent the rest of the day maxiprepping the 1.2 and 1.4 samples and once again failed to yield results. Since I was now out of those samples, we decided to not include sample 1 in the rest of the experiment.

7/27
I selected my clone A cells with 10µL/mL puromycin (500µg/mL) and then infected the 293 cells with DNA from samples 2 and 3 using a Lentivirus protocol.