Rahul Lakhanpal- Third Week

So I wrote this blog over Monday, but then as I went to submit it the internet went out and my whole blog was erased, thus I am forced to rewrite it today (about last week). I have also learned from my mistake and am writing this first on a word document. My third week was a very busy week in which I felt I started to truly move along with my procedure. As I stated in my last blog, my second week concluded with me creating my own primer which we ordered. On Tuesday we received these primers and I set up my first PCR (two containers of DNA). In order to save our DNA and effectively do the PCR we had to dilute our primer. Using the diluted primer I had to use a PCR kit, to get my sample ready for PCR. The PCR kit called for 1 ul of my primer (3-5), 1 ul of another primer (5-3), 1 ul of template DNA, 22 ul of autoclaved h2o and 25 ul of primestar (PCR mix). The PCR then did its cycling over night and the next day I came excited to see my DNA.

In order to confirm if the PCR worked, we had to do a gel electrophoresis. I again got a manual and had to follow steps to first make the gel for the GE, then my solution I was going to put in the wells, and then how to run and read it. The gel was supposed to be 2% agarose which meant I had to measure out 2g of agarose and add  100 ml solution of TAE buffer, and 5 ul of Ethidium Bromide (toxic). I then heated my solution in the microwave for a minute, let it sit, and then poured it into the container in which is solidified into a gel. I then created my two samples in which I was going to load into the wells, they were made of 7 ul of my PCR DNA, 2 ul of h2o and 1 ul of buffer. I loaded them in the wells, along with 2 samples of DNA marker. The loading was at first difficult, but after practice I got pretty good at it. I waited 45 minutes then looked at the gel under a UV light, and it looked pretty well.

But... In the research world a confirmation one time is apparently not good enough… So I had to do another GE on Thursday. I can honestly say, that Thursday was by far the worst day of my young researcher life. On Thursday, none of my GEs were working and I ended up doing 3 trials all fiascos. I left the lab on Thursday at 8pm and was exhausted. The only one more stressed out than me was probably my mother who called me every 20 minutes to make sure I was safe in the city.

After a long Thursday, Dr. Li decided that I should run another PCR so we get better results. So, basically I am pretty much an expert at running both PCR and GE. I set up the PCR reaction and put them in the machine. Next week I look forward to more GEs and along with this Dr. Li also put some cells in to culture and we will be transferring the DNA to them next week! 3 weeks done, onto the next one…

Rahul Lakhanpal

PS: I’m working a short week this week (Monday was July 4, and Tuesday he couldn’t make it back) so I will write my next blog at the end of the week  (hopefully NOT during next week)...