Continuing Left and Tet Overlap and Introducing a New Project

     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 H2O

 

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 H2O Primer Mix  (A & D)

 

We then run these 8 samples on a big 1.2% gel for about an hour. The results of the gel were very disappointing. We had absolutely no DNA on the gel. The pipetting was done very carefully this time, so we know it wasn’t that. Due to this being a reoccurring issue for our group, we are spending our weekend brainstorming possible mistakes that could’ve happened. The gel was laid out as such:

|  L
|
|  #1 - 70°C
|  #2 
|  #3  

|  #4  
|  #5  
|  #6  
|  #7 
|  #8 - 52°C

 

 

 

On another note, I got put on another project today. For the rest of this semester and next semester, Chad and I will be working on his project for his thesis. I currently am not fully informed on the project but getting to work on something other than overlap will be a nice change in scenery!