Week 5

7/9
Today I discovered that, yet again, my bacteria did not grow as I had hoped, so I am starting all over again. I began cloning FSIPPW. Also, I split my clone A cells and EGFP cells onto a six-well dish, with two wells of EGFP at roughly 6x10^5 and three wells of clone A cells at roughly 6x10^5. I also split my 293 cells.

7/10
I went through the CIP treatment, annealing, gel extraction, phosphorylation, ligation, and transformation steps again. However, I began harvesting my virus by inserting the virus into plates of cells, some with Clone A cells and others with EGFP expressing cells.

7/11
Today was another repeat day. I noticed that 2/3 of my bacteria looked great. Sample #2, however, looked questionable. First, however, I added .5mg/ml puromycin to my virus infected cells, and then froze down the extra virus. To tell you the truth, I am getting pretty sick of viruses and how many precautions I must take. The only difference between this virus and my first one is that this one can kill me. Taking this into consideration, I decided to follow proper procedure and soak anything that the virus could have possibly infected in my beaker of bleach. I also got some great pictures of me in my lab coat (though it was way too big on me.)

At the end of the day, I inserted one colony of bacteria into each of 15 tubes, five from each sample. I can only hope that this time they will express the DNA.

7/12
I repeated the entire miniprep day on my bacteria to purify the DNA. I then ran the DNA through an agarose gel, examined it under a UVP light . . . SUCCESS!!! this time I had six out of the 12 colonies (I accidentally contaminated three of my samples) express the segments I had inserted. I picked the three strongest expressing samples and grew them overnight in a 37º high-speed shaker.

7/13
I began the day by splitting my clone A cells, EGFP expressing cells, and 293 cells 1:20—I will be away for a week and don’t want them to overgrow. I then moved my virus expressing cells onto 10 cm plates. I spent the entire day repeating a maxiprep on my bacteria. Every step was performed carefully, double-checking every instruction. Brian told me that if it did not work this time, it would be too late to start over and I would have to find something else to work on. Anticipation was controlling me as I performed my final centrifuge spin of the day. As I removed the supernatant and watched the interior of the tube slowly dry in the open air, I noticed on the bottom of the tubes a clear glob of what I could only hope was purified DNA. I was worried because I did not see this glob on the tube containing what I thought would be DNA sample 1. I was practically sweating as I ran the samples through the photometer. I first ran a blank control first, which printed out as 0.000 µg/ml. Then sample 1, as the machine buzzed and beeped I prayed for good news, only to see a 0.000 µg/ml dsDNA come out of the printer. Now I was scared. As I loaded sample 2 I could sense my project failing . . . but then, success. The printer astonished both me and Brian at the number. 6605 µg/ml dsDNA. He informed me that this was a lot more DNA than he expected and when the third sample printed out a 4670.4 µg/ml I knew that it was no fluke. I happily froze my DNA in the –20º freezer and went home. It turns out that Friday the 13th was my lucky day after all.