Left, Tetracycline, and Right Overlap Update

     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 R₁ isolation was 18.8 ng/μL, and for our R₂ isolation it was 27.7ng/μL. The sample we chose for our overlap reaction was R₂. 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 H20 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 and 35°C. We gel excised these bands by loading the rest of our sample onto a gel. With these samples, we are now attempting to add our left fragment to them. We did four 24μL reactions of 3-fragment overlap. For this 3-fragment overlap, we made sure we had a total of 50ng of right and tetracycline and 40 ng of left fragment. Two of our samples are going in at 55°C and the other two are going to be at 36°C for overlap. The four samples were as follows:

55A – R + T @ 35°C - 3-way overlap @ 54°C 

55B – R + T @ 57°C - 3-way overlap @ 54°C 

36A – R + T @ 35°C - 3-way overlap @ 36°C 

36B – R + T @ 57°C - 3-way overlap @ 36°C 

 

Here are those samples on a gel:

 

 

 

 

 

 

            The sample in the far right of the photo is our 55A sample. Although there is a lot of bands that are nonspecific binding, the band that is right under the rod is our 3-way overlap at 2700bps! Something that is very unfortunate is that when we tried to gel excise it the next day, there was a huge smear. We assumed the reason for that is we loaded way too much of the sample (45μL), which created too much background noise for the band to be evident. Our next steps are to repeat the process that we did with a different gradient. This new gradient is from 64°C-39°C. Also, we are making it so now our fragments will actually be equal molar. Right and Tet will stay at 50ng, but our Left fragment needs to be at 30ng, not 40ng. So this next coming week, we will be doing that gradient, amplifying it, and finally running it on a gel in hopes of recreating those results.