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

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  72C 3min
  72C 3min
  Hold 15C
  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.

Latest revision as of 16:51, 8 September 2014

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

Background[edit]

    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[edit]

  • 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[edit]

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[edit]

  • 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

  • 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

  • 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.