Third Week 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 immediately. After that, centrifuge for 5 minutes to pellet the debris and DNA. From there, you transfer the supernatant (the liquid) to a GeneJET spin column - basically, isolate the pellet. Next, centrifuge again for 1 minute and discard the flow through. Depending on your DNA, you can optionally wash it, then add 500 μl and centrifuge for 1 minute. Then, repeat the wash procedure using 500 μl of the wash solution. After that, transfer the spin column into a fresh microcentrifuge tube. Add 50 μl of Elution Buffer and incubate at room temp for 2 minutes; then centrifuge for 2 minutes. Finally, discard the column and store the purified DNA at -20 degrees Celsius. 



Theory

    On Friday, we waited for the samples of left + tet, right + tet + right, and right fragments to be run under gel electrolysis to see how many base pairs we have. We were expecting to see some bands with around 1700-2700 base pairs depending on which section of DNA we're looking at. Sadly, after running on the gel, we found 0 bands entirely. I don't know why we didn't see any bands, and for a little bit, we thought we may have loaded the incorrect samples, but it just didn't show any readings. That's not to say it didn't run, as the ladder showed up fine, but there were no results on any of the other samples. Another thing I did was work with Deja to figure out the ratios we need for sequencing some more DNA. Basically, we have a bunch of samples of never-sequenced before species, and want to sequence said DNA. We want the concentration of each sample to be 80 μl/mL, so did a bunch of math with the concentration equation (C1V1 = C2V2 ). 

The table of what each sample should contain when we send it off to be sequenced.


Overall, it was sad that we got no results on the gel at the end of the week, but I thin it was overall a very productive week.

Kieran

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