This was working alongside one of the PhD students in the lab, giving me a bit of an experience of what a PhD involves. Looking at it now, I've got to say that it's something that I would be highly interested in doing at some point-- but it is a decision that really shouldn't be taken lightly because it looks like a lot of hard work.
The first day of assisting on this project, I found myself learning the ins and outs of tissue/cell culture. The whole point of the tissue culture is to ensure that at all times there is an established cell line that can be used in the experiments. So I helped out examining the current cultures to see how well they were growing (that's a fancier way of saying that I was counting cells) and sterilizing all the equipment that was being used (as it's all highly open to contamination and when you're trying to grow a cell line this is exactly what you don't want).
After this, I was introduced to the procedures for site-directed mutagenesis-- using PCR. Nick (the PhD student) had designed and had his mutated primers already made, so it simply was just a case of adding the required components and setting the PCR to cycle through the conditions required to cause high numbers of the mutant plasmids to be replicated.
Once the PCR had completely cycled (4 hours later) we then removed the products and discarded the wild type plasmids using a digestion (in which the unmethylated wild type, normal plasmids were removed leaving only the methylated, mutant plasmids behind). We then transformed highly competent E. Coli cells with the plasmids and allowed the cells to incubate overnight.
The following day I was taught how to produce selective agar plates. Normally I would be completely up for doing any practical work-- but unfortunately when you produce agar plates you have to be fully suited and booted to stop any cross-contamination to the plates and you also have to have a bunsen burner going at full whack-- and it was probably one of the warmest days of the summer. Literally so sweltering, I don't think I've been that warm since my tent in Spain.
Anyway, to make the plates a selective medium, we added antibiotics to them which would ensure that only the E.Coli that had taken up our mutants would grow. If the colonies did grow then we'd know that we did, indeed, have a successful mutagenesis... Which we did! (For me that was a little bit of a novelty as I've not exactly had the best results thus far).
Over the next few days we repeated the above procedure several more times for different mutant lines (CIITA Forward, CIITA Reverse, CIITA negative, Zta Forward, Zta Reverse and Zta Negative) which are helping to amass a picture of the protein components of EBV.
Once all of the different mutant cell lines were made, I then took the remaining bacterial cultures and set about extracting the DNA elements from the cells via mini-preps. First for this the cells had to be isolated from their growth medium through centrifugation (the first time I'd ever used a large centrifuge opposed to a table-top one)-- which gave a pellet. The pellet was removed and then resuspended in 2 separate buffers (with Lysate Blu in which gave a colour change to confirm the procedure was being carried out correctly). These solutions were then placed into a table-top centrifuge and spun-- which caused the cellular elements to become debris leaving us with only the soluble components (proteins/RNA/DNA).
After removing the soluble elements I then carried out a separation procedure extremely similar to the procedure I'd used to separate the DNA from agarose a few weeks ago. Having done it before, by the time I'd finished the extraction this time around I felt like a tiny bit of a professional because obviously I could do it at a relatively steady speed without asking for any clarification.
Once the DNA had been purified, I then made a 1 in 10 mastermix of the DNA (diluting it with double-distilled H2O which, I didn't even know existed) which was then analysed via spectrophotometry. This gave us the overall concentration of DNA within the mastermix-- which I then used to calculate making a 50-100ug/ul solution. These resultant solutions have now been sent off for sequencing and they'll hopefully be back at some point this week.
Oh, also at some point over the last week this sign has appeared on our lab wall. It instantly became clear why a career in scientific research is for me:
Anyway, it's hard to believe I'm already halfway through my studentship. It's going extremely fast and before I know it I'm going to be submitting my report (slightly nerve-wracking in itself). Next week I believe I will be returning to my analysis of the OriLyt fragments as my supervisor is back, so I will obviously update to let everyone know what results I'm getting on that front.
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