Chris:LabNotes/FateMapping/Calendar/2014/2014-8-22

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Revision as of 21:18, 22 August 2014 by >Cjwei
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Target capture - Get Rui's probes (positive control) working (Part II - Capture Evaluation prior to full PCR using Phusion)

Background

    rxn     Target (ng)     Probe:Target     Probe (ng)
    1->2    100ng            200:1            3.30 (my probe designed 5/12, on 100ng)
    3->4    100ng           1000:1            1.60 (Rui's probe, on 20ng)
    5       0ng(NTC, CW)     200:1            3.30 (This is NTC for 20hr probe annealing)
    6       0ng(NTC, RL)    1000:1            1.60 (This is NTC for 20hr probe annealing)

Procedure

Component                     1x(ul)     9x(ul)      Init Conc     Final Conc
H2O                          2.6         23.4        --            --
2x Phusion Flash PCR MM       10          90         2x            1x
50x Sybr Green                .4          3.6        50x           1x 
AmpF6.4Sol                     1           9         10uM          .5uM
AmpR6.3.Indx (Indx40)          1           9         10uM          .5uM
Circularized DNA Template      5          --         --            --
                              20
  • 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