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==circularization under different reaction system (10/9/08)== reaction system: 1 2 3 4 5 template + + + - + circleHelper + + + + - Ligase + - + + + Stoffel fragment + + - + + template (509nM) 1ul 10x Ampligase Buffer 2ul CircleHelper V4.2(1uM) 2ul dNTP(10mM) 0.5ul Stoffel fragment(10U/ul) 0.5ul Ampligase(5U/ul) 0.5ul ddH2O 15ul 94C 2 min -> 55C 20 min [[Image:ZhangLab_2 2008-10-09 15hr 52min.jpg]] result: the primer of the first PCR step is not phosphorylated at the 5'end, therefore the ligase would not be able to work. The gel above showed very interesting results, whereas looks like the template and the stoffel fragment are having some unspecific activities. However, I am not entirely sure what happened within each reaction, especially with the 100bp band. The steps will be repeated using phosphorylated primer, and the same reaction system will be used again later to compare the results.
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