Alice:LabNotes/2008-11-24
Jump to navigation
Jump to search
Extend Cosmic Probe[edit]
Spacer insertion template (1) 860nM 1.5 ul spacer mix (100nM each) 20 ul 10x Ampligase Buffer 5 ul Ampligase(5U/ul) 1 ul ddH2O 15.6 ul ---------------------------------------------------------- total 50 ul 95C 5min -> 65C 40min Add 2ul of ExoI&III 37C for 2hrs -> 94C 10min
PCR amplification after spacer insertion system 1 system 2 H2O 35ul 40 SYBG 0.6ul 0.6 primer mix (AP1V4IU+AP2V4) 0.2ul 0.2 Taq master mix 50ul 50 Template (from previous step) 15ul 10 ---------------------------------------------------------------------- total 100ul 100ul
94C 2min -> 94C 45sec -> 58C 45sec -> 72C 30 sec ->plate read -> 20 cycle -> 72C 3min -> 15C TBE gel result: File:ZhangLab 2 2008-11-26 10hr 18min-small.jpg lane 1: PCR result of reaction system 1 lane 2: PCR result of reaction system 2