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===Phusion PCR=== *We are going to use the previous target captured circularlized DNA as template for Phusion reaction *The mastermix used is below (based on the protocol published online [[File:Tech-manual-f-548-phusion-flash-high-fidelity-pcr-master-mix.pdf]]) Component 1x(ul) 20x(ul) Init Conc Final Conc H2O 2.6 52 -- -- 2x Phusion Flash PCR MM 10 200 2x 1x 50x Sybr Green .4 8 50x 1x AmpF6.4Sol 1 20 10uM .5uM AmpR6.3.Indx (Indx40) 1 20 10uM .5uM Circularized DNA Template 5 -- -- -- 20 *Also will include 2 PCR NTC's (two reactions in the third strip) *The two rxn's are PCR NTC (just add 5ul water) *Real-time PCR Profile: (need to change to 98C denaturing) 98C 30sec (98C 10sec -> 58C 30sec -> 72C 20sec)x8 (98C 10sec -> 72C 20sec)x10-15 cycles 72C 3min Hold 15C
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