Linear plasmid construction-8

Creating a linear plasmid is a combination of breaking two different plasmids and attaching them together through different processes. One process in an overlap reaction which consists of primers creating a specific site that both fragments have and willmattach to each other. While another will be restriction enzymes which "cut" the plasmid DNA in a specific way to make either clean cuts or sticky ends. These ends are then attached to each other and put through the PCR machine to see if these a work. Presently I work on the overlap reaction, we have had great success with the left and tet fragments as we can recreate the band needed for this project with these fragments multiple times.

 There was some inconsistencies with the combination of outright fragment from deinococcus redidurens and the tet fragment from e.coli. The most we can see so far is that the combination of the fragment causes some kind of complex to bunch up and not leave the wells. As such we are testing to see if there is a heat of the Pcr machine that we could have. Running a multitude of heat gradients such as a 30*C-50*C and then a combination leading to the highest being 67*C through the final heat gradient. We are waiting to see if Restriction enzymeswill yield the same results or if the fragments of right and tet are just not specific enough, if that is the case then a change of primers would be needed. 

This is a gel of a heat gradient of tet and right, one at 37*C and another at 57*c

This gel is an older sample of right and tet that was run for a comparison of present DNA

This gel is the 5μl of sample that shows we do have some 3 way assembly even though it was filled with other DNA and interference.