Chris:LabNotes/FateMapping/Calendar/2014/2014-9-5: Difference between revisions

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>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...")
 
>Cjwei
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**AmpLigase Addition: (add 2ul into rxn's depending on specified times below)
**AmpLigase Addition: (add 2ul into rxn's depending on specified times below)
  Component            Stock conc    Final conc    1x(ul)    MM Vol (18x) (ul)     
  Component            Stock conc    Final conc    1x(ul)    MM Vol (18x) (ul)     
  AmpLigase            5U/ul          0.5U/ul        0.4       7.2           
  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          
  10x AmpLigase Buff  10x            1x            0.2        3.6
H2O                                                1.6      28.8   
*Below are the steps for each individual reaction:
*Below are the steps for each individual reaction:
  1) Add 2ul Hemo KlenTaq solution into each at start of 4hr incubation
  1) Add 2ul Hemo KlenTaq solution into each at start of 4hr incubation

Revision as of 16:31, 5 September 2014

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        0.2        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