Kasper Kasabach- Week 2

The second week was just as eventful, if not more, than the first.  This week was mainly focused on new and more difficult techniques as well as follow specific and arduous protocols.  Monday, Tuesday, and Wednesday, I worked on a maxiprep with Kris to help me prepare to do it on my own.  The purpose of a maxiprep is to prepare the plasmid and insert it into the bacteria.  The first step in the process is to grow the bacteria.  To do this, the cells with the new plasmid need to be placed into a beaker with the optimal conditions for them to survive which includes SOC media.  This type of media has all of the necessary nutrients to allow bacteria to flourish.

The next part of maxiprep is to wash and then use lysis buffer to lyse the cells, allowing the plasmid with the desired gene, in this case a certain cancer resistance gene, to be harvested and eventually tested in other environemnts.  

The final portion of maxiprep is focused on purifying the plasmid.  This part is done using DNA binding columns and various different buffers including QC, QBT, and elution buffer.  The first two buffers are used to clean the bacteria, and the third is used to take away the DNA.  The DNA then dissolves the appropriate amount in the elution buffer. 

Once we were finished with the Maxi, we moved onto doing a miniprep.  The miniprep serves the same purpose as the maxiprep does, but it just goes about purifying the plasmid differently.  This technique uses PE buffer instead of the QC or QBT buffers.  It also uses restriction enzymes such as ECO-RI and ECO-RV to cut the plasmid in the appropriate place in order to insert the DNA.  

On Thursday, Kris wasn't in the lab because she was moving.  This day I worked with Olga instead, on a completely different project.  Her project consisted on developing new cell lines to be used in other experiments.  This new cell line of CARs needed to have a specific antigen on the surface.  The way this was done was by combining the 12 flasks of cells into two larger flasks.  A sample was then taken out of each flask and counted to see what percent of the cells were alive and how many cells there were total.  There were about 2 billion cells in the solution, with about 86% remaining alive.  These flasks were then spun down and the supernatant was removed.  Wash buffer was then added and the supernant was again removed from the flasks, this time via vacuum.  New freezing and R10 media were then added and the solution was frozen in 24 cryotubes with 1mL in each.  

Friday was a completely different day.  The first three and a half hours were spent at a lab safety training session which focused on hazardous lab precautions and how to monitor the safety of all of the members of a lab.  There were over 150 people there and 41 were PIs.

After this, we went out to our EXP lunch were we met up with other people and got to here about the amazing work that everyone is doing in the lab.  It was very enjoyable and relaxing.  Almost everyone at the table ordered a burger.  After this, Dr. Peretz came to my lab and met my lab supervisor.  Unfortunately my PI wasn't there at the time, but the visit went well.  By four, almost everyone left my lab for the weekend.