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==Circularization== Equation: final volume x final concentration = initial volume x initial concentration the final concentration of template is 20nM, so 20nM x 30ul = 956nM x amount we need, which is 0.623 ul. CircleHelper V_short is 100uM, so we take 1 ul of it and add 99ul of H2O to dilute it 100 times to 1uM. The final concentration of circlehelper is 60nM, so we need 1.8ul. Add the ligase at 0.02 units/ul ratio. since the total volume is 30ul, we will need to add 0.12 ul of ampligase. T4 ligase is 2000U/ul. so we will dilute it to 5U/ul, which is to add 0.5ul of ligase to 199.5ul of H2O. reaction system 1: template (956nM) 0.6ul 10x Ampligase Buffer 3ul CircleHelper V_short(1uM) 1.8ul Ampligase(5U/ul) 0.12ul ddH2O 27ul ----------------------------------------- total 30ul 94C 2 min -> 55C 20 min reaction system 2: template (956nM) 0.6ul 10x T4 ligase Buffer 3ul CircleHelper V_short(1uM) 1.8ul T4 ligase(5U/ul) 0.12ul ddH2O 27ul ----------------------------------------- total 30ul 94C 2 min -> 25C 20 min add 2ul Exo I &III 37C 2hr -> 95 10min -> 4C Purify using Minelute columns TBU gel result: [[Image:ZhangLab_2 2008-10-15 15hr 32min.jpg]]
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