Chris:LabNotes/FateMapping/Calendar/2014/2014-8-19

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Target capture with padlock probes (designed 5/12) test with Rui's probes as control (Part II - Capture Evaluation prior to full PCR using Phusion)[edit]

Background[edit]

    rxn     Target (ng)     Probe:Target     Probe (ng)
    1       100ng            500:1            8.25 (my probe designed 5/12, on 100ng)
    2       100ng            200:1            3.30 (my probe designed 5/12, on 100ng)
    3       100ng             50:1            0.83 (my probe designed 5/12, on 100ng)
    4       100ng           1000:1            0.32 (Rui's probe, on 20ng)
    5       0ng(NTC, CW)     500:1            8.25 (This is NTC for 20hr probe annealing)
    6       0ng(NTC, CW)     200:1            3.30 (This is NTC for 20hr probe annealing)
    7       0ng(NTC, CW)      50:1            0.83 (This is NTC for 20hr probe annealing)
    8       0ng(NTC, RL)    1000:1            0.32 (This is NTC for 20hr probe annealing)
    9       0ng(NTC)        ----              0    (This is full NTC, no template and no probe)
---------------------------------------------------------------------------------------------------------------------------------------------------
    10      0ng(NTC, CW)     500:1            8.25 (This is NTC for the 4hr gap-filling and circularization step, to be added AFTER annealing step)
    11      0ng(NTC, RL)    1000:1            0.32 (This is NTC for the 4hr gap-filling and circularization step, to be added AFTER annealing step)     
---------------------------------------------------------------------------------------------------------------------------------------------------
    12      0ng(NTC, CW)     500:1            8.25 (This is NTC for Exo digestion step, to be added AFTER gap-filling and circularization)
    13      0ng(NTC, RL)    1000:1            0.32 (This is NTC for Exo digestion step, to be added AFTER gap-filling and circularization)

Procedure[edit]

Component                     1x(ul)     18x(ul)     Init Conc     Final Conc
H2O                          2.6         46.8         --            --
2x Phusion Flash PCR MM       10         180         2x            1x
50x Sybr Green                .4         7.2         50x           1x 
AmpF6.4Sol                     1          18         10uM          .5uM
AmpR6.3.Indx (Indx40)          1          18         10uM          .5uM
Circularized DNA Template      5          --         --            --
                              20
  • 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 are the gel images from the PCR result:

File:Cw ZhangLab 2 2014-08-19 18hr 41min pp capture test phusion 1.jpg

File:Cw ZhangLab 2 2014-08-19 18hr 42min pp capture test phusion 2.jpg