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=Target Capture - Try Different Extension/Ligation Times (Part II)= ==Background== *This procedure is a continuation of probe capture from <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-9-4> *As a reminder, below is a description of the reactions we're going to be testing: rxn Target (ng) Probe:Target Probe (ng) Description ------------------------Strip A (CW probes)------------------------ 1 100ng 200:1 3.30 Hemo KlenTaq + AmpLigase 4hr (control) 2 "" "" "" Hemo KlenTaq 4hr + AmpLigase 2hr 3 "" "" "" Hemo KlenTaq 4hr + AmpLigase 1hr 4 "" "" "" Hemo KlenTaq 4hr + AmpLigase 0.5hr 5 0ng -- 3.30 Hemo KlenTaq + AmpLigase 4hr (control) 6 "" "" "" Hemo KlenTaq 4hr + AmpLigase 2hr 7 "" "" "" Hemo KlenTaq 4hr + AmpLigase 1hr 8 "" "" "" Hemo KlenTaq 4hr + AmpLigase 0.5hr ------------------------Strip B (RL probes)------------------------ 1 100ng 1000:1 1.60 Hemo KlenTaq + AmpLigase 4hr (control) 2 "" "" "" Hemo KlenTaq 4hr + AmpLigase 2hr 3 "" "" "" Hemo KlenTaq 4hr + AmpLigase 1hr 4 "" "" "" Hemo KlenTaq 4hr + AmpLigase 0.5hr 5 0ng -- 1.60 Hemo KlenTaq + AmpLigase 4hr (control) 6 "" "" "" Hemo KlenTaq 4hr + AmpLigase 2hr 7 "" "" "" Hemo KlenTaq 4hr + AmpLigase 1hr 8 "" "" "" Hemo KlenTaq 4hr + AmpLigase 0.5hr ==Procedure== *Because we'll be adding Hemo KlenTaq and AmpLigase separately, we'll need to make the two solutions separately and add them at the specified times (as a reminder, we already have 20ul in tube): **Also note that we're using a different dNTP mix (instead of using dNTP from Epicentre Phi-29 kit [25mM each], we'll be using the one from NEB [10mM each]) **Hemo KlenTaq Mastermix: (add 2ul into each rxn at the beginning of the 4hr incubation step) Component Stock conc Final conc 1x(ul) MM Vol (18x) (ul) Hemo KlenTaq 100% 20% 0.4 7.2 dNTP 1000uM 100uM 0.2 3.6 (NEB dNTP = 10mM, so do 1/10d) 10x AmpLigase Buff 10x 1x 0.2 3.6 H2O 1.2 21.6 **AmpLigase Addition: (add 2ul into rxn's depending on specified times below) Component Stock conc Final conc 1x(ul) MM Vol (18x) (ul) AmpLigase 5U/ul 0.5U/ul 0.2 3.6 (Want to keep number of units added to rxn the same [i.e. 1U]) 10x AmpLigase Buff 10x 1x 0.2 3.6 H2O 1.6 28.8 *Below are the steps for each individual reaction: 1) Add 2ul Hemo KlenTaq solution into each at start of 4hr incubation 2) Add 2ul AmpLigase solution to tubes '''1,4''' (for strip A/B) at start of 4hr incubation 3) Add 2ul AmpLigase solution to tubes '''2,5''' (for strip A/B) at 2hr mark 4) Add 2ul AmpLigase solution to tubes '''3,6''' (for strip A/B) at 3hr mark 5) Add 2ul AmpLigase solution to tubes '''4,8''' (for strip A/B) at 3.5hr mark *Thermocycler (continued): Add 2ul SLN to each rxn -> '''Incubate 60C 4hr''' -> 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) 20x(ul) Init Conc Final Conc H2O 2.6 52 -- -- 2x Phusion Flash PCR MM 10 200 2x 1x 50x Sybr Green .4 8 50x 1x AmpF6.4Sol 1 20 10uM .5uM AmpR6.3.Indx (Indx40) 1 20 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 ==Results== *Below is the gel image for strip A (CW probes):(L,1->8) [[File:cw_ZhangLab_2_2014-09-05_19hr_27min_ligase_time_test_phusion_gel_A.jpg|500px]] *Below is the gel image for strip B (RL probes): (8->1,L) [[File:cw_ZhangLab_2_2014-09-05_19hr_29min_ligase_time_test_phusion_gel_B.jpg|500px]] *Seems like positive control didn't work. Not quite sure why (this is a new tube of Rui's v6 probes so maybe something is different with the tube? Or perhaps the reagent concentrations were off because of the two step AmpLigase/Hemo KlenTaq? Also, I used the new NEB dNTP for this run, so maybe try using the old Epicentre dNTP?) Will repeat just the positive control next week.
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