Deja Vu Birney's TRAIN Blog

     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 10 μL 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

  For our experiment, we are attempting to combining the left fragments of   deinococcus radiodurans and  and our fragment of tetracycline, taken from E. coli, with our right fragment of   D. rad.  The last time we combined and amplified our fragments, we used the Anna protocol and Hilgarth protocol. On 10/5 we ran a gel on the result of that PCR overlap and amplification and ended up with nothing. No DNA was shown in our samples. We believed that it could be due to bad master mix. For this weeks portion of our project, we focused on doing one slightly different protocol (Reaction  #1) and repeating our Hilgarth protocol (Reaction #2).     For reaction #1 we ran our 2 samples of left, right, and tet. fragments through PCR with primers 1, 4, 5, and 6. The recipe used for our PCR is below: Reaction #1- Total volume: 50µL   11µL Master Mix 2µL DNA 1µL Primer 1 1µL Primer 2 1µL Primer 3 1µL Primer 4 3µL PCR      The reason for adding these primers is to pinpoint our DNA outside of the over