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...

  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...

    This week in lab, I learned how to make and load a gel. For this blog, I will be discussing the protocol on how to make a 30mL gel. Before starting the process of making my gel, I had to figure out what percent of agarose we should use. For my practice gel, I did 1% agarose (percentage determined by how small or large the DNA is that you're using). For my conversion, I did the following equation: 30mL  *  0.01  *  1000  =  300mg             (percent  (conversion              agarose)    to mg)      I measured 30mL of TAE buffer in a flask a little over twice the volume. I then measured 300mg of agarose on my scale, added it to my buffer, then swirled until cloudy. Before putting my gel solution into the microwave, I made a stopper out of paper towel. After inserting the stopper, I put the solution in the microwave at 30 second increments until i...

      This week during lab, we were working on cleaning up left and right fragments. We are cleaning the sample of everything except the DNA that we want. The protocol I learned for this cleanup is as follows: We diluted the left and right samples with the DNA Cleanup Binding Buffer according to a 5:1 ratio recommended for fragments. We then loaded sample into a column that was within a collection tube and spun for 1 min at 13,000 rpm. We then proceeded to discard the flow through. After we re-inserted the column in another collection tube, adding 200µL of DNA Wash Buffer and spun again for 1 minute. We repeated the last step once more before transferring the column into a 1.5µL microfuge tube. Finally we added 10µL of DNA Elution Buffer to our samples, ensuring that its going into the center of the matrix. Then, we waited for 1 minute before proceeding to spin it for another 1 minute at 13,000 rpm. After our first elution cycle, we put the samples on the nano drop, the r...

    This week during lab we completed a gram stain on Deinococcus radiodurans after it was grown in MHA (mueller-hinton) media. Deinococcus radiodurans is an extremophile bacteria that we are growing in our lab. We are trying to grow  Deinococcus radiodurans  for 24 hours, in a media that we've never had in before, to see if it's successful.       To complete my gram stain, I first started by drawing a wax circle on my slide for and an indicator in the top right corner, to ensure my slide wouldn't be upside down at any time. I then ignited my Bunsen burner then sterilized my loop with the flame. After allowing the loop to cool, I unscrewed my tube containing the  D. rad  in MHA media, ran the top of the tube over the flame, collected my culture, ran the mouth of my tube over the flame once again, before finally screwing my tube shut. After closing tube, I smeared my culture within the wax circle on my slide. I waited about 2 minut...