Attempting Overlap with Anna and Hilgarth Protocol

  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 overlap. Primers 1 and 4 will create a band on our gel where our left and tet. combined fragments are (1700 bp). Primers 4 and 5 will give us a band on our gel where our right fragment would be (1000 bp). This will show us if our DNA is still good. Ideally what we would want to see on this gel are those two bands I described previously, along with a third band at 3700 bp. This band would confirm that some of the DNA in our sample did in fact overlap, creating a left, right, and tet. fragment. 

    For reaction #2, we are doing step three of the Hilgarth protocol. For the Hilgarth protocol we diluted primers 1 and 6 using a ratio of 1:10 (1µL of primer to 10µL PCR water). We also did touchdown starting at 60ºC. We are redoing this protocol because we believe that the master mix was a factor on why nothing showed up on our gel during our last reaction. The recipe we used for PCR is below:

Reaction #2

22.5µL Master Mix

4 µL DNA

1 µL Diluted Primer 1

1 µL Diluted Primer 6

21.5 µL PCR Water

 

    Another reaction we are putting through PCR and running on a gel is lactonase. We are using lactonase as a control in this experiment to help us determine if it’s the DNA that is bad or other components. If we see lactonase is showing up on our gel and our other samples aren’t, we can conclude that it is the DNA that is causing our lack of results. If we are seeing no bands from all three reactions, then we believe it has to do with our master mix. At the end of this blog, I added a pdf of the Hilgarth protocol we have been using in our experiment.