Kasper Kasabach- Week 3

This week was a little different than the previous two.  Since July 4th was on a Tuesday this year, the lab had off both Monday and Tuesday.  A few international members of the lab went to the lab anyway on Monday, but not Tuesday.  I decided to spend these first two days of the week going to the beach with my family.  We had a great time down there.


After returning from the beach on Tuesday we went down to Penn's landing to go see the fireworks in the evening.  

Image result for penn's landing fireworks 2017 july 4th

Finally the work began.  This week was full of mini and maxipreping with bacteria.  These bacteria were One Shot Top 10 bacteria that had a plasmid insertion.  The Y2F plasmid was inserted into these bacteria to grow and multiply.  A minprep was then preformed on these bacteria to make sure that the plasmid was really there.  We then ran these plasmids on a gel.  One of them was fully digested to make sure that the plasmid was the correct one.  When the bands did appear in the proper places on the gel (about 350 base pairs long), we used a gel extraction kit to cut out and purify the Y2F portion which was then inserted into two different types of CARs, CARHER2 and CAR19.  We next needed to prepare these two different CARs by growing them in a solution of SOC media and LB.  





This experiment was not effective in growing the amount of plasmid needed, so we had to spend another two days of the week growing new bacteria.  This time, we used Stabl3 bacteria because they are known to grow better.  They did grow better, but the transformation percentage was much lower with these cells.  Luckily, there were so many more cells that it didn't matter.  These were first grown in LB media again overnight.  Next they were concentrated and selected for on a Ampicillin plate overnight.  This time, many well dispersed colonies grew on the plate, making it usable.  At this point it was Friday and the bacteria needed to be spun down the next day or they would die.  So, I had to come in that Saturday to come spin down and freeze the bacteria over the weekend. This wasn't super complicated but it felt like I was the only person in the building, it was eerie.

Image result for sorvall centrifuge

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