Practicals

 Introductions: pRAD1

    For this week's research, we focused mainly on practical experience. Getting in the lab and doing some hands-on learning. We began our research by using pRAD1 which contains a promoter, Amp R, Ori, and CmR; it is also resistant to ampicillin and cloriphenical. We started with our e.coli broth from the previous week, which we resuspended with the vortex since it was sitting for a week. After the broth has been resuspended we then removed 1 mL of the broth and placed it into an Eppendorf tube. Following the broth being added to the Eppendorf tube, we then centrifuged it for 30 seconds at 13,000 rpm. The end goal was to lice the cells so we can get plasmid. After the tube is done in the centrifuge we discarded the supernate (liquid) and kept the tiny pellet. 


     After we discarded the supernate we resuspended the pellet in a B1 buffer solution of 200 uL. We then added the B2 buffer solution to our Eppendorf tube. Gently inverting the tube we mixed the solutions together and then incubated at room temperature for 1 minute. Then add an additional 400 uL of our B3 solution, then incubate again. After the incubation time was up, the Eppendorf tube was placed in the centrifuge for 5 minutes. After the 5 minutes were up, we removed the supernate again. 



    The supernate alone was then placed into the centrifuge. While the supernate was spinning we heated out E solution to 50 degrees celsius. 


    Finally, we use 200 uL of wash buffer 1 to the plasmid, then place it back into the centrifuge for another minute. Discard the supernate. Add 400 uL of wash buffer 2 to the plasmid, as well as 30 uL of E solution we were previously warming up. Place back into the centrifuge for an additional minute. And the little liquid remaining at the bottom is hopefully plasmid. 


After we get the tiny liquid at the bottom, it is time to test it on the Nano-Drop

Nano-Drop

    We first use ethanol to clean the nano-drop, making sure all our tests are accurate. Use 2 uL of elute as a blank (baseline). Make sure to place elute on the tiny bulb. Press blank to zero out the nano-drop. Then place 2 uL of the sample (plasmid) onto the bulb. For my test, I got the results: 

128.0 ng/uL

1st 1.83 A260/A280

2nd 1.83 A260/A280

Comments

  1. EricaMarch 3, 2022 at 12:42 PM

    Nice work, but maybe take a moment to touch on the theory behind the procedure. Why is there a precipitation step, etc.

    ReplyDelete

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