Chris:LabNotes/FateMapping/Calendar/2014/2014-9-5
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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