Plasmid DNA Transformation Lab Report

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Due to limited time constraints and having lab only once a week this lab was broken up into five periods, the last one specifically to go over the results. Day one consisted of extraction of plasmid DNA, which we used Resuspension Buffer to break up our pellet after removing the supernatant (liquid) after centrifuging our overnight culture. Then Lysis Buffer was used to break down the membrane of the cells so that we could then add Neutralization Buffer to bring our sample from basic to neutral pH. After centrifuging a few times with some steps in between we added Wash Buffer to remove all other liquid from the spin column leaving just the DNA in its membrane. Later some Elution Buffer was used to separate the DNA from the membrane of the spin column. Day two consisted of making the plasmid DNA resistant to Kanamycin and Ampicillin. Day three we made 0.8% agarose gel and ran electrophoresis to be able to see the weight of the DNA. Day four we genetically transferred the ligated plasmid DNA to E.coli and plated the cell mixture. Day five we looked at our results and went over them. All of this works together for bacterial transformation, we transformed E.coli to be Kanamycin and Ampicillin resistant, by adding the Kanamycin and Ampicillin plasmid DNA to E.coli.
Procedure:
Start this lab by isolating plasmid DNA from E.coli so that we can use it in recombinant DNA. First we placed 1mL of overnight culture into a microcentrifuge tube, then centrifuged it for five minutes at full speed. Once it was done, we removed the supernatant careful to not disturb the pellet. Next we added 250µl of Resuspension Buffer and pipetted it up and down till the pellet could no longer be seen. Then 250µl of Lysis Buffer was added to the tube we closed the cover and inverted the tube six times, then it had to sit for four minutes. After four minutes we added 350µl of Neutralization Buffer and repeated the steps we just did for the Lysis Buffer. Once the four minutes were up we centrifuged our tube for ten minutes so that the DNA was left in the supernatant and the cell debris was in a pellet. Next we carefully removed all the supernatant that we could and placed it in the top of a spin column in a new microcentrifuge tube. Then it was centrifuged for one minute, the liquid the made it through the membrane of the spin column was disposed of. After the tube was emptied we placed the spin column back on top we washed it by adding 750µl of Wash Buffer and centrifuging it again for one minute. Once again we discarded the liquid and then places the tube back under the spin column. Then centrifuged the tube and spin column for one minute. Next the spin column was placed in a new microcentrifuge tube and 50µl of Elution Buffer was added to the top of the column and it sat for one minute before centrifuging for a minute. The liquid in the tube now contained our plasmid DNA and the spin column was discarded. Next we labeled two tubes and put 5µl of our DNA into each of them. Tube one we also added 8µl of 2x Reaction Buffer, 1 µl of EcoRI, and 1µl of HindIII. Tube two we also added 8µl of 2x Reaction Buffer and 2µl of H2O. Once the tubes were made we put them into the microcentrifuge quickly just to get all the liquid to the bottom of the tubes. Next the tubes were placed into a float and put into a cold water bath set to 37ºC. The next thing we did was make
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First we ran a gel electrophoresis to make sure that we had obtained the DNA like we thought. Once the electrophoresis was done we could clearly see that we did have DNA (Figure 1). Then we added the DNA to the ligated E.coli, once this was done we added the samples to a plates that had Kanamycin and Ampicillin in the agarose to see if we were at all successful in making the E.coli also resistant to Kanamycin and Ampicillin. Looking the plates we can see many colonies grew, so we were again successful (figure

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