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===Phusion PCR=== *We are going to use the target captured circularized DNA as template for Phusion reaction *'''Note: Since we are not going to do qPCR, we don't need to add SYBR into rxn mix''' *Mastermix recipe (we'll need to decrease the PCR volume because of the higher number of reactions required and limited reagents we have on-hand; to make things simple, we'll use the same input template volume into each PCR reaction) Component 1x(ul) 100x(ul) Init Conc Final Conc H2O 10 1000 -- -- 2x Phusion Flash PCR MM 25 2500 2x 1x AmpF6.4Sol 2.5 250 10uM .5uM AmpR6.3.Indx 2.5 (add separately) 10uM .5uM Circularized DNA Template 10 (add separately) -- -- 50 *Below are the indices used for the reactions: (this is based on the spreadsheet <https://docs.google.com/spreadsheets/d/1F2L2NpqYCXYwYPk1WQdJDkj3yZKMJftYfeYUHtiXCL4/pubhtml#>; Indices ) **The following file (bottom table) contains information on the pooling and indices used for the different reactions: [[File:cw_20150812_PhuPCR_index-seqs.pdf]] [[File:cw_20151023_PhuPCR_barcodes.pdf]] *Thermocycler Profile: (need to change to 98C denaturing) 98C 30sec (98C 10sec -> 58C 30sec -> 72C 20sec)x8 (98C 10sec -> 72C 20sec)x'''12 cycles''' 72C 3min Hold 15C
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