Linear plasmid

 This week was done by using a different protocol. The protocols were then named the ANNA protocol and the HIlgarth protocol. The Hilgarth protocol includes a new procedure called a touchdown, which is when the PCR machine will slowly lowers the temperature of the reaction. This touchdown was done on different samples of our left fragment of deinococcus and the tetracycline fragment from E.coli. To do this two different samples went through the ANNA at 37*c and the Hilgarth at 72*c, then each of these samples was run through the step 3 final fusion of the Hilgarth protocol. The fragments of left and tet were not as successful as we thought they would be. As the left fragment reacted very well to the new touchdown protocol, tet did not. So the fragment of left and tet that was already created went through an ANNA protocol and another sample of left and tet went through the Hilgarth protocol. Step3 of the Hilgarth protocol needed diluted primers from 10x stock to 1x. So taking 1μl of the 10x stock and 9μl, except I did add 10μl instead. These fragments were then run on a gel, on this gel, there was exactly nothing, no streak nothing. Following this 4 tubes were put on a gel 2 were run through ANNA and HIlgarth separately with no diluted promes or touchdown, and 2 tubes were run through Anne and HIlgarth separately with diluted primes and touchdown. The undiluted primers were, 1,4,5,6 primes were added in volumes of 2.5μl. The diluted primes were done with 10μl of primer and 90μl of DI Water. 

Figure 1: This is the gel run with the left fragment and tet fragment (which was found to be insufficient) 

Our observations of these different protocols show that making a linear plasmid with overlap is not easy and have to keep trying. The gel shows that while the left fragment reacted well to the touchdown protocol, the tet fragment did not. Also when adding all of the fragments together has not worked so far, we are still hopeful that the Hilgarth protocal will show results or maybe a mixture of both methods.