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

From ZhangLabWiki
Revision as of 16:19, 5 September 2014 by >Cjwei (Created page with "=Target Capture - Try Different Extension/Ligation Times (Part II)= ==Background== *This procedure is a continuation of probe capture from <http://genome-tech.ucsd.edu/LabNote...")
(diff) ← Older revision | Latest revision (diff) | Newer revision → (diff)
Jump to navigation Jump to search

Target Capture - Try Different Extension/Ligation Times (Part II)

Background

    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        0.4        7.2            
10x AmpLigase Buff   10x            1x             0.2        3.6            
  • 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