Kasper Kasabach- Week 6

This week was all about making viruses.  I needed to make a Virus using the CAR19 + Y2F plasmid as well as 3 other viruses.  The process of making viruses is initially very complicated, but toward the end of the process gets much clearer and simpler.  There are a ton of reagents that need to be mixed with the plasmid DNA and the 293 cells.  This cell line is used because it is one of the easiest to transduce.  The process of making the Lente Virus began by mixing the plasmid DNA, 293 cells, and reagents that allowed for the plasmid to more easily transduce the cells.  These cells were then put in an incubated flask at 37 degrees overnight with 60mL of media.

The next day, the media had been almost completely drained by the cells and was now filled with the desired viruses.  We removed this media, collecting it in 2 50mL conical tubes and added a fresh 60mL of media to the flasks so that they could continue producing more virus.  These were put back in the incubator.  The 50mL conicals were then spun down at 1000 x g for 10 minutes in order to bring all of the debris to the bottom.  The virus samples were then filtered to eliminate the last bits of debris from the sample.  These samples then needed to be put in the ultracentrifuge and spun at 10,000 x g for 24 hours.  The next day, these samples were removed and the virus was resuspended in 1mL of media rather than the 29 that it had been in during the spin.  These tubes were then spun down at 25,000 x g for two and a half hours.  The samples were again resuspended in even less media and then transferrered into cryotubes to be frozen.

The next day, the exact same process was done to extract more virus from the cells.  At this point, viruses 4 was the only one that could last until 72hrs, so the cells were left to keep growing.  The other three viruses were then collected and the cells were discarded.  The same process was done with these viruses as the day before.  More virus was frozen so that it could be titled and tested next week.
This was also the week that I had to give my lab presentation about everything that I had been working on since beginning in the lab in June.  I brought in donuts and we had a very relaxed lab meeting.  My presentation was about 45 minutes, and after, everyone hung around the conference room and talked for a while.

Time went by so fast in the lab this summer.  I had a great time and would really like to thank everyone that helped me out, in particular Saar, Kris, and Mike.