Chris:LabNotes/FateMapping/Calendar/2014/2014-8-22: Difference between revisions

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=Target capture - Get Rui's probes (positive control) working (Part II - Capture Evaluation prior to full PCR using Phusion)=
=Target capture - Get Rui's probes (positive control) working (Part II - Capture Evaluation prior to full PCR using Phusion)=
==Background==
==Background==
*Continue from
*Continue from <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-8-21>
*Below is the experimental design again:
    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==
*We are going to use the previous target captured circularlized DNA as template for Phusion reaction (Phusion found in common reagents box in -20C)
*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)    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

Revision as of 21:18, 22 August 2014

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