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==Procedure== ===Prep KLN mix solution ('''Strips A and C''')=== *We are using HemloKlenTaq for the reaction (because AmpliTaq Stoffel was discontinued) *First, perform the following dilutions: **Dilute dNTP 1/10d (2ul dNTP + 18ul H2O) (10mM/nt -> 1mM/nt) *Mastermix recipe Component Stock conc Final conc 1x(ul) MM Vol (18x) (ul) Hemo KlenTaq 100% 20% 0.4 7.2 AmpLigase 5U/ul 0.5U/ul 0.2 3.6 dNTP 1mM 100uM 0.2 3.6 10x AmpLigase Buff 10x 1x 0.2 3.6 H2O 1.0 18 *Aliquot 4.5ul KLN mix to each well of a strip tube so can use multichannel when adding to the reactions *Add 2ul KLN mix to '''Strips A and C''' rxns 1->8 using multichannel ===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''' ===ExoI/ExoIII treatment=== *We want to then add 20U ExoI and 100U ExoIII *Mastermix recipe Component Stock Conc Final Amount 1x(ul) MM Vol (34x) (ul) ExoI 20U/ul 20U 1 34 ExoIII 200U/ul 100U 0.5 17 Water 0.5 17 *Aliquot 8.5ul 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 **Because Hemo KlenTaq and Phusion have different reaction volumes (24ul vs 42ul), we'll need to set up two different PCR reactions for each. The reason why is because we want to keep the input amount of DNA approximately the same (i.e. input half of the volume of the reactions for PCR) *'''Note: Since we are not going to do qPCR, we don't need to add SYBR into rxn mix''' *Mastermix recipe (Strip A, 24ul reaction from capture) Component 1x(ul) 16.5x(ul) Init Conc Final Conc H2O 28 462 -- -- 2x Phusion Flash PCR MM 50 825 2x 1x AmpF6.4Sol 5 82.5 10uM .5uM AmpR6.3.Indx 5 (add separately) 10uM .5uM Circularized DNA Template 12 (add separately) -- -- 100 *Mastermix recipe (Strip B, 42ul reaction from capture) Component 1x(ul) 16.5x(ul) Init Conc Final Conc H2O 19 313.5 -- -- 2x Phusion Flash PCR MM 50 825 2x 1x AmpF6.4Sol 5 82.5 10uM .5uM AmpR6.3.Indx 5 (add separately) 10uM .5uM Circularized DNA Template 21 (add separately) -- -- 100 *Also include one PCR NTC using the mastermix made for strip B *Below are the indices used for the reactions: (this is based on the spreadsheet <https://docs.google.com/spreadsheets/d/1F2L2NpqYCXYwYPk1WQdJDkj3yZKMJftYfeYUHtiXCL4/pubhtml#>) Strip A rxn Indx Indx Seq (RC) 1 20 CCTGTC 2 21 CAACCC 3 22 CTCGGA 4 23 TCGAAA 5 24 ATTCGC 6 25 TACTGC 7 26 TCGTGA 8 19 GACGCA <- This overlaps with the PCR NTC from 2015_03_04 probe capture ------------------- Strip B rxn Indx Indx Seq (RC) 1 27 TACGCG 2 28 AGGTGC 3 29 ACGAAC 4 30 TTAGTG 5 31 CACCAC 6 33 CGCAAC <- Skip Index 32 because was identified as bad and not sure if they changed the sequence for these primers yet 7 34 AACTGA 8 19 GACGCA <- This overlaps with the PCR NTC from 2015_03_04 probe capture ------------------- Strip C rxn Indx Indx Seq (RC) 1 35 ATCGGG 2 36 CAAGCA 3 37 GCTACA 4 38 TCACGC 5 39 CCAGAG 6 40 TGACGG 7 41 GGGAAC 8 19 GACGCA <- This overlaps with the PCR NTC from 2015_03_04 probe capture ------------------- Strip D rxn Indx Indx Seq (RC) 1 42 GAAAAG 2 43 AGTGCC 3 44 GCATCC 4 45 ACTACG 5 46 CATTTC 6 47 CTCTCC 7 48 ACGTTA 8 19 GACGCA <- This overlaps with the PCR NTC from 2015_03_04 probe capture *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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