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

From ZhangLabWiki
Jump to navigation Jump to search
>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
 
(3 intermediate revisions by the same user not shown)
Line 33: Line 33:
**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
Line 44: Line 45:
**Note: Right before 4hr is up, prep the ExoI/ExoIII mix at 1:1 ratio
**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
**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.

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.