Chris:LabNotes/FateMapping/Calendar/2015/2015-10-23
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Target Capture - Using C1 amplicons and extended MDA from WZTO CBL) w/ 9/22/2014 probes (Hemo and Phusion Reactions) (Part II)[edit]
Background[edit]
- This is a continuation of the experiment started on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-10-22>
- As a reminder, the reactions for this experiment are described in the file below (40 C1 amplicons in total + 2NTC, all with 9/22/14 probes; one set with Hemo and another with Phusion)
File:Cw 20151022 C1 WZTO CBL.pdf 1 2 3 4 5 6 7 8 9 10 11 12 A ---------------------------------1------------------------------------ B | C V D ---------C1 Amplicons----- 2NTC ---------C1 Amplicons---------- 2NTC E | F | G V H ---------------------------------8------------------------------------- ----------Hemo-------------------|-----------------Phusion-------------
Procedure[edit]
Prep KLN mix solution (6 strips, 8 rxn's each)[edit]
- 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)[edit]
- 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[edit]
- 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[edit]
- 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)x12 cycles 72C 3min Hold 15C