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=Target Capture - Using C1 amplicons and extended MDA from 2015_08_10 C1 run (WZTO BA9, P44G BA9) w/ 9/22/2014 probes (Hemo and Phusion Reactions) (Part II)= ==Background== *This is a continuation of the experiment started on <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2015/2015-8-11> *As a reminder, the reactions for this experiment are described below (46 C1 amplicons in total + 2NTC, all with 9/22/14 probes; one set with Hemo and another with Phusion) 1 2 3 4 5 6 7 8 9 10 11 12 A ---------------------------------1------------------------------------ B | C V D A1 A2 A3 B1 B2 B3 A1 A2 A3 B1 B2 B3 E | F | G V H ---------------------------------8------------------------------------- ----------Hemo-------------------|-----------------Phusion------------- ==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 (108x) (ul) ExoI 20U/ul 20U 1 108 ExoIII 200U/ul 100U 0.5 54 Water 0.5 54 *Aliquot 26ul 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 '''Make two of these in order for total of 52 rxns''' 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#>) **<span style="color:red">'''Note: We are overlaping indices for single cells across the two different enzymes (when combining the different libraries, make sure combine in equal amounts based on gel quantification; sequence the two enzyme conditions in two separate lanes in HiSeq RapidRun). However, we want to make sure that the indices for each individual single cell amplicon is different'''</span> **The following file contains information on the pooling and indices used for the different reactions: [[File:cw_20150812_PhuPCR_index-seqs.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 ==Results== *Below are the gel images from the capture done in this experiment: [[File:cw_ZhangLab_2_2015-08-13_10hr_48min_PPCap_Gel_1A-H,2A-D.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_10hr_50min_PPCap_Gel_2E-H,_3A-H.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_10hr_52min_PPCap_Gel_4A-H,5A-D.jpg|200px]] (Note that one of the lanes showed nothing because loading volume was very low...probably pipetting error) [[File:cw_ZhangLab_2_2015-08-13_10hr_54min_PPCap_Gel_5E-H,6A-H.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_12hr_01min_PPCap_Gel_7A-H,8A-D.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_12hr_02min_PPCap_Gel_8E-H,9A-H.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_12hr_04min_PPCap_Gel_10A-H,11A-D.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_12hr_05min_PPCap_Gel_11E-H,12A-H.jpg|200px]] *Note that while pooling and size selecting, I needed to do Qiaquick purification in order for the gel to look okay (otherwise the bands are not clear due to the high amounts of salt). Otherwise, the gels looked fine as shown below: [[File:cw_ZhangLab_2_2015-08-13_17hr_13min_PPCap_pre-size_selection_L,pool1-4.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_17hr_15min_PPCap_pre-size_selection_pool5,L,pool6-8.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_17hr_30min_PPCap_post-size_selection_L,pool1-4.jpg|200px]] [[File:cw_ZhangLab_2_2015-08-13_17hr_31min_PPCap_post-size_selection_pool5,L,pool6-8.jpg|200px]]
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