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===Prep Phusion/AmpLigase mix solution ('''Strips B and D''')=== *We are using the Phusion/AmpLigase mix described in the paper *First, perform the following dilutions: **Dilute dNTP 1/10d (2ul dNTP + 18ul H2O) = use same diluted dNTP as above **Dilute NAD+ 1/10d (15ul NAD+ + 135ul H2O)(50mM -> 5mM) *Mastermix recipe Component Stock conc Final Amount 1x(ul) MM Vol (18x) (ul) NAD+ 5mM 40nmol 8.0 (after dil) 144 dNTP 1mM 600pmol 0.6 (after dil) 10.8 Betaine 5M 15umol 3.0 54 10x AmpLigase Buff 10x 1x 2.0 36 AmpLigase 5U/ul 10U 2.0 36 Phusion HF DNA Pol 2000U/ml 6.4U 3.2 57.6 Water 1.2 21.6 *Aliquot 45ul Phusion/AmpLigase mix to each well of a strip tube so can use multichannel when adding to the reactions *Add 20ul Phusion/AmpLigase mix to '''Strips B and D''' rxns 1->8 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'''
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