Deja Vu Birney's TRAIN Blog

       These past couple weeks in lab have been very interesting. First, we decided to give the right fragment of   deinococcus radiodurans   and tetracycline from   E. coli  a try. We are doing this because we haven’t seen much success in adding our right fragment to our left and tet fragments. We initially had isolated more right fragment via gel excision. We had performed two different right isolations. Our ng/μL reading on R 1   isolation was 18.8 ng/μL, and for our R 2   isolation it was 27.7ng/μL. The sample we chose for our overlap reaction was R 2 . We then decided to do an 8-sample overlap heat gradient of right fragment and tetracycline from 35 °C to 60°C. The recipe we used is below:   11μL 7μL 2μL 4μL  Master Mix PCR H 2 0 Right Fragment Tetracycline Fragment             From L to R: 60°C, 57.9°C, 54.6°C, 50.1°C, 44.5°C, 39.6°C, 36.7°C, 35°C   The two best bands we saw were at 57°C...

       Last week in lab we had run our left fragment from   Deinococcus radiodurans,   and our tetracycline fragment from   E. coli.  On a heat gradient from 34 ° C to 54 ° C. This gradient led to our discovery of the ideal temperature for a fragment overlap, that being 54 ° C. With this, we decided to play around and do another gradient. This gradient was from 52 ° C to 70 ° C with a total of 8 reactions. We did have to make more left and tetracycline fragments as we were running low, so we had spent most of our time amplifying those samples and gel excising them. For our overlap reactions, we used the recipe below:   5µL 7µL 11µL 1µL Left Tet Master Mix PCR H 2 O   We ran these 8 samples through PCR in their gradient. We then amplified the samples using the recipe below:   5µL 10.75µL 2.25µL 2µL Overlap Sample Master Mix PCR H 2 O Primer Mix  (A & D)   We then run these 8 samples on a big 1.2% gel for about an hour. ...

This week, we did a PCR overlap heat gradient ranging from 34°C to 54°C on our left fragment of  deinococcus radiodurans  and our tetracyline fragment from  E. coli.  When we ran this reaction on a gel, there was absolutely no DNA showing up. We assumed there was maybe a problem with the pipetting of the fragments into the tubes when they were going through PCR for amplification, and we decided that it would be best to start from the beginning again to ensure that there was no error. We then proceeded to redo the overlap as well as the amplification process at 34°C to 54°C. For these reactions, we made sure they were equal molar. We first had to dilute our samples down to left being 10ng/µL and tet being around 5ng/µL. We did this by getting our original samples and adding PCR water. With our goal of having a 50ng/µL total for left and a 35ng/µL for tet, we loaded the amount below.   5µL 7µL 11µL 1µL Left Tet Master Mix PCR H 2 O   With this ...

     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...