Week 2

6/18
I began the week by taking another look at my death curve in case anything changed over the weekend. Luckily, the results remained unchanged and I was now certain of the optimal ratio of puromycin to use. Then, I began my first cell count. A cell count consists of using simple ratios to count the approximate number of cells in a culture plate. First, I resuspended the cells on my plate into 8mL of growth media. Then I removed 10µL of the media and mixed it with 10µL of special stain. I took 10µL of this stain and spread it onto a glass plate, which contained a grid pattern. Using a microscope, I counted the number of cells on four of the grids on the plate and ended up with the number 191. I multiplied 191 by 2 (a 1:1 dilution) and then divided the result (382) by 4 (four grids). Because of the way the glass plate is designed, this meant there were 95.5 x 104 cells (950,000) in 1mL of media. Because there was roughly 7.5mL of media, I multiplied my number by 7.5 and concluded that there were 7,100,000 cells in the media. I then resuspended my cells in 7.1mL of media, so that each mL of media should contain 1 million cells.

The special RNA I ordered came in today, and that allowed me to begin work on the second phase of my project. I started to clone an FSIPPW DNA backbone, first by digesting it. I added 8.34µL of FSIPPW, 25.66µL H20, 4µL EcoRI Buffer, 1µL EcoRI, and 1µL BamHI. I placed the backbone and enzymes in a 37 degree water bath overnight.

I also resuspended 293-T cells in FT media and left it to incubate overnight at 37 degrees

Then I suspended the special oligos I ordered in RNAse-free water so the concentration was .1nM

6/19
This morning I added 1µL of CIP to digest the FSIPPW mixture. I incubated that at 37 degrees for 1 hour. Then I added 8µL of EDTA to inactivate the CIP, and left that at 75 degrees for 10 minutes. I ran the mixture through a 1% agarose gel with a 1kbp DNA marker. I later extracted a glowing band (visible with UV light) and eluted the pure DNA with a Qiagen gel extraction kit (this took forever).

I annealed the oligos I suspended the day before with a PCR program called Liz oligo, which consists of 95 degrees for 10 minutes, 70 degrees for 10 minutes, decreasing 1 degree/minute until it reaches 25 degrees. Phosphorylating these oligos consisted of adding the oligos to T4PNK and ATP, which I incubated at 37 degrees for 30 minutes, then 70 degrees for 10 minutes, then put on ice. I then began the Ligation process, adding the oligos to the purified FSIPPW and ligase, with a negative control that used water instead of the Oligos.

Transformation consisted of preparing LB media by adding MgC12, MgSO4, and glucose. I mixed 3.4µL B-ME per 200 µL aliquot XL-1 blue competent cells into a 1.5 mL tube. Then, after adding 1 µL of the ligation mix to each tube, I incubated them on ice for 30 minutes, heat shocked them at 42 degrees for 90 seconds, and then put them back on ice for 2 minutes before adding the cell mixture to 1 mL of warmed media and shaking at 37 degrees for 45 minutes. I then resuspended in 100µL of media and spread on a LB/Amp plate.

I froze one of my plates of 293-T cells in a cryogenic freezer (liquid nitrogen tank) and then split my other stock onto a plate and added G418 “neomycin.”

With my clone A cells, I added polybrene to the 6-well plate, then added the retrovirus. One thing I did learn about working with viruses, however, is that people are paranoid of what they can do. I worked with a retrovirus today, like HIV/AIDS, but the only effects are a slight immunity to neomycin and glowing green under a certain light. I had to have a beaker of bleach next to me at all times, and everything that touched what could have virus on it had to be rinsed and then ejected into bleach. The end result was a beaker filled more with pipet tips than with the bleach itself. After this, I rinsed everything with bleach and then put the cells in the incubator.

6/20
Oh, man…
The colonies did not grow on the LB/Amp plates, so I repeated the transformation step of yesterday’s experiment, adding twice the amount of cells to the plates as last time. Also, instead of shaking the bacteria for 45 minutes, I left them in there for three hours so they had more time to recover. I then began digesting another batch of FSIPPW in case the E. Coli still did not grow.

I removed the virus from the cell plate and replaced the media with regular growth media, then placed the cells back in the incubator.

Since today was a slow day, I helped Brian with some clone A cells of his. I added different secondary stains to his cells, some receiving Fascin 1:50, others Fascin 1:100, a third group Mouse IgG, and a fourth Phalloidin. Then I proceeded to perform a long and rather annoying wash-rinse-repeat cycle using TBS-T and PBS.

6/21
Bad news for me . . .
The colonies still did not grow, so I repeated all of the experiments that I did on the 19th. Then I mounted cover slips with Brian on the cells I had rinsed yesterday.

6/22
Finally!
The E. Coli had grown, so I picked five of the best colonies from each tray and isolated them in their own media. I then put them in a 37 degree high-speed shaker overnight. Then I went back to helping Brian.

I performed a task known as rtPCR with a Sox 9 protein. To make a really long story short, we performed a PCR on the protein and later, examining the gel, we found a band around where we wanted one to be, so I wasn’t really sure what that meant but he seemed pretty clear that it was good news.

I counted and split my 293 cells, then replaced the media in the virus/control cells with 1µg/mL G418. Finally, I split my clone A cells and went home for the weekend.