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Cell Lysing

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Cell Lysing  Over the past few weeks, I've been learning how to lyse cells with my lab partner, Alex. We started with a paper (Kikuchi) on lysing Deinococcus radiodurans - then using the information from it's protocol to try and lyse cells that are  Deinococcus aquaticus .  Here's the protocol we pulled from the paper -  MATERIALS AND METHODS D. radiodurans MR., KR, (I1) and Sark were used in this experi-ment. One milliliter of overnight culture was inoculated into 200 ml of TGY medium (1). The bacterial cells were harvested after cultivation from 24 to 36 h at 30°C. The cells were washed twice with Multibuffer (50 mM sucrose, 10 mM Tris-HCI, 40 mM EDTA, 0.1% Triton X-100, pH 8.0) and were lysed by the incubation with lysozyme (1 mg/ml) for 5 min at 37°C. Plasmids were isolated by the alkaline lysis method (12). For the analysis of plasmids, agarose gel electrophoresis was carried out using Tris-acetate buffer (12) at 100 V constant voltage. Two agarose concentration...
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Fourth Week Blog

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 Fourth Week Fun     This week, we ran a second large gel holding a different set of samples from last week. We used the separate set hoping that it would show up on the gel, as the samples the previous week didn't show on the gel at all. After running the gel, not even primers showed up, which is why we ran the second set of samples, hoping that we would get results. We added 10.75  μl   of mastermix, .5  μl  FWD, .5  μl  DNA, and 37.75  μl of PCR water. After loading the results on a gel, we let it run for about 2 hours. After running the gel again we got the same results as the previous week, however, it's very possible no primers we added to the samples. Another thing I did was learn more about enzyme restriction/ligation from Lizzy and Jonathon, and how they might change their protocol to help with the DNA's binding.  New Protocol for Enzyme Restriction/Ligation      1. Measure the DNA concentration of inser...
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Third Week Blog

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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...
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Second Week Blog

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...
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First Week Blog

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...
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