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

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Target Capture - Troubleshoot v6 probes (Part II)[edit]

Background[edit]

1) v6 probes, all-in-one KLN mastermix with old Epicentre dNTP     rxn's 1,5
2) v6 probes, all-in-one KLN mastermix with new NEB dNTP           rxn's 2,6
3) v6 probes, separate KLN mastermix with old Epicentre dNTP       rxn's 3,7
4) v6 probes, separate KLN mastermix with new NEB dNTP             rxn's 4,8

Procedure[edit]

  • Prep two different dNTP dilutions:
    • Epicentre MDA dNTP = 1/25d
    • NEB dNTP = 1/10d

Rxn's 1,5 (all-in-one KLN, old Epicentre dNTP), 2ul added[edit]

Component            Stock conc     Final conc     1x(ul)     MM Vol (5x) (ul)     
Hemo KlenTaq         100%           20%            0.4        2.0            
AmpLigase            5U/ul          0.5U/ul        0.2        1.0            
dNTP                 1000uM         100uM          0.2        1.0       (MDA dNTP = 25mM, so do 1/25d)     
10x AmpLigase Buff   10x            1x             0.2        1.0            
H2O                                                1.0        5           

Rxn's 2,6 (all-in-one KLN, new NEB dNTP), 2ul added[edit]

Component            Stock conc     Final conc     1x(ul)     MM Vol (5x) (ul)     
Hemo KlenTaq         100%           20%            0.4        2.0            
AmpLigase            5U/ul          0.5U/ul        0.2        1.0            
dNTP                 1000uM         100uM          0.2        1.0       (NEB dNTP = 10mM, so do 1/10d)     
10x AmpLigase Buff   10x            1x             0.2        1.0            
H2O                                                1.0        5           

Rxn's 3,7 (separate KLN, old Epicentre dNTP), 4ul added[edit]

Component            Stock conc     Final conc     1x(ul)     MM Vol (5x) (ul)     
Hemo KlenTaq         100%           10%            0.4        2.0            
dNTP                 1000uM         50uM           0.2        1.0       (MDA dNTP = 25mM, so do 1/25d)     
10x AmpLigase Buff   10x            1x             0.4        2.0            
H2O                                                2.8       14.0          
AmpLigase            5U/ul          0.25U/ul       0.2        1.0  

Rxn's 4,8 (separate KLN, new NEB dNTP), 4ul added[edit]

Component            Stock conc     Final conc     1x(ul)     MM Vol (3x) (ul)     
Hemo KlenTaq         100%           10%            0.4        2.0            
dNTP                 1000uM         50uM           0.2        1.0       (NEB dNTP = 10mM, so do 1/10d)     
10x AmpLigase Buff   10x            1x             0.4        2.0            
H2O                                                2.8       14.0          
AmpLigase            5U/ul          0.25U/ul       0.2        1.0 
  • Thermocycler (continued): Add 2ul or 4ul KLN to each rxn -> Incubate 60C 4.5hr -> 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)    11x(ul)      Init Conc     Final Conc
H2O                          2.6           33        --            --
2x Phusion Flash PCR MM       10          110        2x            1x
AmpF6.4Sol                     1           11        10uM          .5uM
AmpR6.3.Indx (Indx40)          1           11        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 this experiment
    • The gel lanes are as follows:
x     L     1->10 (where 9,10 are PCR NTC)

File:Cw ZhangLab 2 2014-09-09 17hr 53min positive control troubleshoot.jpg

  • Looks like the positive control worked for all conditions. Perhaps it was just contamination by accidentally adding in my probes instead of v6 probes? This time around, I did do repeat pipetting whenever I added in the new mixes, so I'll make sure to continue doing that for next time.
  • For now, I'll try either repeating the AmpLigase/Hemo KlenTaq in separate steps experiment again or try with the new Taq fragment that Dinh gave me or with Phusion instead of Hemo KlenTaq. However, first, I'll regroup and look into the mappability/alignability of capture arms as Dr. Zhang suggested.