S-STEM Scholar Kieran Younger's Blog

Third Week Wonders     This week, I mostly worked with gels and PCR again. I worked with Lani's group on Tuesday to amplify left + tet. We wanted a 10.75 MM with 2 μl of DNA, 1  μl primer (x2 for both ends of the DNA), and filled the rest with PCR water to reach a total of 20 μl. While the PCR went on, I also helped with making some media for other bio classes. We started by adding a small glass tube into a second vial, then filling it about halfway with solution.  The first gel run on Tuesday. Gave exactly the readings we hoped for.  Plasmid Extraction     While I haven't done a plasmid extraction by myself yet, I can still go over the protocol and what it entails. The first step is to resuspend the pellet in 250 μl of resuspension solution. Mix it all together, then centrifuge it. Next, we add 250 μl of Lysis solution. You should mix well by inverting the tube 4-6 times. Next, you add 350 μl of the neutralization solution and mix immed...

Week 2 Daily Dues     This week, I personally made a gel and loaded it, and learned more about enzyme restriction/ligation, overlap, plasmid extraction, and PCR. At the beginning of the week, we started growing more e. coli with tet and did some plasmid extractions to get the tet from the e. coli. We also started the Hilgarth and Anna(I don't happen to have the Anna protocol ) reactions over again to figure out which reaction worked better for us. I didn't do any of the reactions myself, so I'll go over what I did learn.  PCR     PCR is the process of making more of a certain section of DNA. You do it through three steps, the first being denaturing, the second annealing, and the third step extension. The first step, denaturing, starts by breaking down the DNA. It does this by breaking the bond of the double helix, and sort of unwinding it. You start this step at 94-95 degrees Celsius to break it down, but change the temperature at the second step, annealing...

First Week Learning      Thi s week , I learned about PCR, gel electrolysis, overlap, and restriction enzymes. We did some amplified some left + tet, and tried to combine the left + tet with the right. Despite the group’s best efforts, we were unsuccessful with getting a good sample of left + tet + right. While the experiments were happening, however, I was busy learning how to do said experiments - so I’ll go over what I leaned.  Gel Electrolysis       I learned how to make a gel to read how many base pairs a sequence of DNA has. First, you figure out how dense you want the gel, based on what DNA you’re trying to read. Afterwards, you make the gel by combining agarose and TAE, boiling it for 5 minutes, then let it harden from a liquid to a  gel while you have a comb inserted in it to make wells. After the gel is hardened, you can load your sample(s) of DNA into the wells - loading the first or second well with your ladder. The ladder is used...