Kasper Kasabach- Week 4-5

Week 4 and 5 were mainly focused on the same thing, transforming bacteria.  Since last week, the Y2F plasmid was not cut properly, yielding many bands of different sizes, rather than a band around 6k and one at 350 base pairs long.  We first double checked the plasmid map to make sure that the enzymes that we used were in fact the correct ones.  They were, so we contacted the company about the improper cuts.  They did their own tests and found that the enzymes were manufactured improperly.  They remade the construct and sent it back.  This time, the proper bands appeared.  We then had to send these bands out for sequencing to make sure that the inserts were in fact the mutated form of the Y2F insert, since the mutant and wt versions are the same length.  The image below shows how sequencing is done.

The sequencing for the CAR19 with a Y2F insert came back with the results that we expected, the bands matched the mutated version exactly.  From here, we needed a larger colony of bacteria, so they were plated and grown over night so that there were hundreds of colonies.  These colonies were then used for a maxiprep which purified the plasmid DNA out of these cells.  These cells did not grow super well in the LB, but there were enough to harvest the plasmid.  We expected a concentration of DNA of about 1ng/ul, however the concentration was actually .725ng/ul.  As long as the concentration is above .5ng/ul, it is usable to make virus, which as done during week 6.  

The other thing that I did this week was Harvest more macrophages, but this time from human samples.  This much different than with the mice because the samples came in bags from the hospital.  The first thing that needed to be done with these bags is to scrape them against the cabinet to dislodge the macrophages, a sticky type of cell.  The bags were then emptied and refilled with media using a special syringe.  The process was then repeated, but this time with acutase to try to further dislodge any of the remaining cells.  The process was fairly successful, yielding about 70 million macrophages.    

The last big thing that I did during these two weeks is learning how to use the Fortessa or flow cytometer.  This machine is extremely complicated and would take me months of practice to become proficient at using it, but I learned the basics anyway.  The purpose of the Fortessa is to preform all different types of useful assays to monitor and analyze cell growth, expression, and many other chemical and physical properties.  The way it works is buy shooting a laser through your stained samples to emit light of different ewavelengths on the other side.  These are then captured and expressed on graphs depicting data for the assay that you were using.