Overlap

 Overlap PCR

This was a week of overlapped PCR reactions. For this project to overlap, PCR protocol was needed, as such, there were buffers and calculations needed. For our sample, there was about 100ng/μl, so we took only 2μl per solution so which would give a total of 50ng/μl. From there a master mix was made that contain ruffly  5x buffer- 5μl, 5x high concentration GC buffer- 5μl,dNTP's- 0.5μl,Q5 polymerase- 0.5μl , PCR water- 13.5μl. This was put through 15 cycles in the PCR machine without any primers, hopefully sticking together the Left fragment of D.Rad and of tetronomycin with its promoters. To get the 50ng/μl wanted taking 3μl of the sample and 3μl of the elution buffer following taking 1μl for the PCR reaction, this reaction was run for 15 cycles at 36*C. Afterward was put through a regular PCR reaction which is a reaction that needed 50μl total with primers attached. 

Figure 1; Shows a picture of a slow run gell of the fragments left and tetromycin 

Figure 2; a picture of a gell run (from left to right) ladder, cleaned gell, PCR cleanup

From these pictures, it can be shown that we do in fact have a tet and left fragment connected, what is not clear is why figure 2 is so dirty. Both of the gel fragments should have come out almost as clean as the first one. Since they did not a few questions are asked. Does the PCR and Gel cleanup kits need to be checked? Did the fragments not connect? Why is there so much genetic carryover?