Plasmid Extraction

    This week we started with running a couple gels for our different fragments. I worked on the left fragment, which unfortunately did not give the results we were hoping for. Since it was all the same sample, just ran on different temperatures in the thermal-cycler, we combined them all and will work later on cleaning them up. 



   We took our e.coli broth and used that as our base. Originally we started with only 1mL of our sample, and after the plasmid extraction was completed, there was very little DNA in the sample. We then used 5mL to extract enough DNA to work with. We started with centrifuging 1mL at a time and discarding the supernate. After we did 5 different cycles of centrifuging we had our pellet



    Once we had the pellet we resuspended it with 200 micro-liters of our B1 buffer. Then added an additional 200 micro-liters of the B2 buffer (Lysis Buffer), followed by gently inverting the sample until it was mixed. An additional 400 micro-liters of B3 buffer (Neutralizing Buffer) was added, then gently inverted till the same turned a uniform yellow. Once the sample was yellow it was placed inside the centrifuge for 5 minutes. After the 5 minutes were up, the supernate was then placed inside special tubes with filters at the bottom. With the supernate being in the new tube we then centrifuged again, this time for 1 minute. Then followed up with 200 micro-liters of wash buffer 1, and placed back inside the centrifuge for an additional minute. After the minute was up 400 micro-liters of wash buffer 2 was added, and then the sample was centrifuged again. Finally, we switched our filter tube to a tube with a lid and then added 30 micro-liters of our elution buffer, then placed it back inside the centrifuge for its final spin. 

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