Doing Gel Excision on Left, Tet, and Right Fragments After Anna Protocol

     This week, we performed gel excision and extracted our left and right fragments of deinococcus radiodurans and our tetracycline fragments from our gel that was run after we did PCR with our Anna protocol. We excised the piece of gel with the DNA. We did this by putting our sample on UV light and cut out our bands. For the next step, we added 3 volumes of our Gel Dissolving Buffer. We did 3 volumes instead of 2 because our gel slices were > 150mg. We then incubated our gel pieces at 55°C, inverting periodically, for about 10 minutes. After, we inserted a column into a collection tube, loaded the sample, then spun it down, discarding flow through. After, we added DNA wash buffer into our column, spun it down, and repeated once more. For the final steps, we added the column to a clean microfuge tube and added DNA Elution Buffer. We added 10μL of our Elution Buffer to our sample, then spun it down. After nano dropping our samples, here are out results:

Left 9.1 ng/μL     1.95 A280     0.35 A230

Tet 11.5 ng/μL    1.88 A280     0.77 A230

Right 5.6 ng/μL      1.93 A280     0.03 A230

    With these results, we went ahead and attempted overlap with out left and tet fragments. For this overlap, we need to make sure all of our fragments are equal molar. We did 8 reactions of left and tet on a gradient of 44°C to 34°C in our PCR. I don't have a picture of the results, but our most ideal temperature for our fragments are around 44°C. To double check that the temperature is ideal at 44°C, we are going to do a gradient from 34°C to 54°C.

Comments

  1. Keep in mind you need to cite your sources and these blogs should represent 9 hours of work each week.

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