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==Procedure== *Prep two different dNTP dilutions: **Epicentre MDA dNTP = 1/25d **NEB dNTP = 1/10d ====Rxn's 1,5 (all-in-one KLN, old Epicentre dNTP), 2ul added==== Component Stock conc Final conc 1x(ul) MM Vol (5x) (ul) Hemo KlenTaq 100% 20% 0.4 2.0 AmpLigase 5U/ul 0.5U/ul 0.2 1.0 dNTP 1000uM 100uM 0.2 1.0 ('''MDA dNTP = 25mM, so do 1/25d''') 10x AmpLigase Buff 10x 1x 0.2 1.0 H2O 1.0 5 ====Rxn's 2,6 (all-in-one KLN, new NEB dNTP), 2ul added==== Component Stock conc Final conc 1x(ul) MM Vol (5x) (ul) Hemo KlenTaq 100% 20% 0.4 2.0 AmpLigase 5U/ul 0.5U/ul 0.2 1.0 dNTP 1000uM 100uM 0.2 1.0 ('''NEB dNTP = 10mM, so do 1/10d''') 10x AmpLigase Buff 10x 1x 0.2 1.0 H2O 1.0 5 ====Rxn's 3,7 (separate KLN, old Epicentre dNTP), 4ul added==== Component Stock conc Final conc 1x(ul) MM Vol (5x) (ul) Hemo KlenTaq 100% '''10%''' 0.4 2.0 dNTP 1000uM '''50uM''' 0.2 1.0 ('''MDA dNTP = 25mM, so do 1/25d''') 10x AmpLigase Buff 10x 1x 0.4 2.0 H2O 2.8 14.0 AmpLigase 5U/ul '''0.25U/ul''' 0.2 1.0 ====Rxn's 4,8 (separate KLN, new NEB dNTP), 4ul added==== Component Stock conc Final conc 1x(ul) MM Vol (3x) (ul) Hemo KlenTaq 100% '''10%''' 0.4 2.0 dNTP 1000uM '''50uM''' 0.2 1.0 ('''NEB dNTP = 10mM, so do 1/10d''') 10x AmpLigase Buff 10x 1x 0.4 2.0 H2O 2.8 14.0 AmpLigase 5U/ul '''0.25U/ul''' 0.2 1.0 *Thermocycler (continued): Add 2ul or 4ul KLN to each rxn -> '''Incubate 60C 4.5hr''' -> 94C 2min -> add 2ul ExoI/ExoIII mix (1:1) to each rxn -> 37C 2hr -> 94C 5min -> Hold 10C **Note: Right before 4hr is up, prep the ExoI/ExoIII mix at 1:1 ratio **Note: Made sure to equilibrate to 37C for at least 1min before adding in Exo ===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) 11x(ul) Init Conc Final Conc H2O 2.6 33 -- -- 2x Phusion Flash PCR MM 10 110 2x 1x AmpF6.4Sol 1 11 10uM .5uM AmpR6.3.Indx (Indx40) 1 11 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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