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==Procedure== ===Prep KLN mix solution (6 strips, 8 rxn's each)=== *We are using HemloKlenTaq for the reaction (because AmpliTaq Stoffel was discontinued) *First, perform the following dilutions: **Dilute dNTP 1/10d (6ul dNTP + 54ul H2O) (10mM/nt -> 1mM/nt) *Mastermix recipe Component Stock conc Final conc 1x(ul) MM Vol (54x) (ul) Hemo KlenTaq 100% 20% 0.4 21.6 AmpLigase 5U/ul 0.5U/ul 0.2 10.8 dNTP 1mM 100uM 0.2 10.8 10x AmpLigase Buff 10x 1x 0.2 10.8 H2O 1.0 54 *Aliquot 13ul KLN mix to each well of a strip tube so can use multichannel when adding to the reactions *Add 2ul KLN mix to all strips (columsn 1->6) using multichannel ===Prep Phusion mix solution (6 strips, 8 rxn's each)=== *First, perform the following dilutions: **Dilute dNTP 1/10d (6ul dNTP + 54ul H2O) (10mM/nt -> 1mM/nt) (same as above) **Dilute NAD+ 1/10d (45ul NAD+ + 405ul H2O)(50mM -> 5mM) *Mastermix recipe Component Stock conc Final Amount 1x(ul) MM Vol (53x) (ul) NAD+ 5mM 40nmol 8.0 (after dil) 424 dNTP 1mM 600pmol 0.6 (after dil) 31.8 Betaine 5M 15umol 3.0 159 10x AmpLigase Buff 10x 1x 2.0 106 AmpLigase 5U/ul 10U 2.0 106 Phusion HF DNA Pol 2000U/ml 6.4U 3.2 169.6 Water 1.2 63.6 *Aliquot 130 Phusion mix to each well of a strip tube so can use multichannel when adding to the reactions *Add 20ul Phusion mix to all strips (columns 7->12) using multichannel *Thermocycler (continued): Add KLN or Phusion mix to each rxn -> '''Incubate 60C 4hr''' -> 94C 2min **'''Note: I took out tubes immediately after 94C incubation was done and put on ice for minute then add Exo mix and vortex/centrifuge then put back in thermocycler for 37C 2hr incubation''' ===ExoI/ExoIII treatment=== *We want to then add 20U ExoI and 100U ExoIII *Mastermix recipe Component Stock Conc Final Amount 1x(ul) MM Vol (116x) (ul) ExoI 20U/ul 20U 1 116 ExoIII 200U/ul 100U 0.5 58 Water 0.5 58 *Aliquot 28ul Exo mix to each well of a strip tube so can use multichannel when adding to the reactions *Thermocycler (continued): Add '''2ul ExoI/ExoIII mix''' -> 37C 2hr -> 95C 5min ===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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