As I said in my last blog in order to verify that the ligation was successful, highly
competent bacterial cells were transformed. Bacterial cells were grown on LB
Ampicillin plates, and incubated overnight. There were two types of colonies
visible on the plates 24 hours post transformation that differed in size and
brightness, so I assumed the transformation was successful. Random colonies were selected for a colony PCR, which would verify that the bacteria was transformed. I designed primers to amplify a 1200 basepair fragment that would indicate correct orientation
if colonies harbored the plasmid. Results from gel electrophoresis verified
that a colony harbored the plasmid. This selected colony and three random
others were minipreped and sent for DNA sequencing. The sequence had 7 mutations, as I compared it to one on NCBI. Looking at a DNA waveform without DSAP was not challenging, because I was very comfortable doing this. My lab was very surprised that I knew what a waveform even was, and were thrilled that I could do this tedious task by myself. I constructed a glycerol stock using the colony that harbored the plasmid. A glycerol stock is a way to store bacteria for later use, so for this particular experiment it is useful. I did a maxi prep of the colony. A maxi prep is a very tedious, full day task. However, this day was broken up into a wonderful visit by Dr.Peretz. Dr. Peretz got to see my awesome lab and meet my even more awesome PI. I am hoping Dr. Mason can find time in her very very busy schedule to fit us in! It was nice to share my work with somebody outside of the lab. We also went to the cutest restaurant ever!! The maxi prep yielded 2,500 nanograms
of DNA, so my lab was excited!! Human stem cells were transfected on a 6 well plate with turbofact and
liptofectamine 2000. Turbofect Transfection Reagent is recommended for transfection of plasmid DNA. While most transfection reagents are lipid-based formulas like Lipofectamine2000, turbofact is a sterile solution of a proprietary catonic polymer in water. The polymer forms positively-charged complexes with DNA that are both compact and stable. These complexes protect DNA from degradation and facilitate efficient plasmid delivery into eukaryotic cells. 24 hours post transfection, cells were transfected and
glowing green from GFP protein in the vector. About 70% of cells were
transfected. Cells were prepared and stained for flow cytometry, which with various controls would conclude if CD21 was expressed on the surface of the cells. I saw a slight shift in staining, which concluded that CD21 was probably expressed on the surface but not in an immense amount. The lab is now going to continue to work on this gene due to my success. Overall, I feel like these 6 weeks in the lab taught me a lot of life skills (taking trains and such), and also proved my abilities to work in a lab. I know I had a very solid project, that has many promising applications, and I completed it mostly alone. I know I helped the lab out a lot this summer, whether it be a miniprep here or there or saving time for others. My last day at the lab was extremely bittersweet. My lab took me out to lunch at a very fun mexican restaurant, and wrote me a very meaningful card. I gave gifts and cards to everyone in the lab who has been so great to me, and it was sad for everyone to see me go. I am welcome back to work at the Mason lab in the future anytime, and will definitely miss everyone and my work. I feel confident that I was productive in the lab, and that I took advantage of this amazing opportunity from peddie. As for continuing a career in research a PhD student I spent time with at the lab wrote to me, "You still have a long while to go before you make any decisions (and then decide again after getting a different degree), and there is no need to rush. You certainly seem comfortable in the lab, and have what we call "good hands" - a knack for things going well at the bench - but I imagine that you would be an asset wherever you go and whatever you decide to do. But, if you do decide to go into lab research, you can be sure that the Mason lab will be cheering you on. I cannot say that it will be easy or even fun all the time, but it will definitely be an adventure." On that note, have a good summer everybody <3
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