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Alice:LabNotes/2008-7-28
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==<b>Insert Spacer into Circularized DNA (8/17/08):</b>== The concentration of cosmic probe after Alu-1 digestion is 23.7 ng/ul, which is 897 nM. The volume will be added into the reaction will be 5ul, which makes it 4.485 umol. 107bp spacer concentration is 155.5 ng/ul, which is 3.001 uM. We need to achieve 1:1 probe/spacer ratio, so the volume of spacer needed is 1.495 ul. 299bp spacer concentration is 237.8 ng/ul, which is 2.019 uM. The volume of spacer needed is 2.221 ul. template 5ul template 5ul spacer(100bp) 1.5ul spacer(300bp) 2.2ul 10x Ampligase Buffer 2.5ul 10x Ampligase Buffer 2.8ul 94C 3min -> 60C 20mins -> add 1ul SLN mix -> 60C 40mins -> add 2ul Exo I/III-> 37C 2h -> 94C 5mins >4C ddH2O Ligase Buffer Ligase Amplitaq dNTP 2U/ul AmpliTaq Stoffel fragment; 0.5U/ul AmpLigase; 0.2mM dNTP: 0.52ul 0.1ul 0.1ul 0.2ul (2.5mM)0.08ul x20 10.4ul 2ul 2ul 4ul 1.6ul
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