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

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Target capture - Get Rui's probes (positive control) working (Part II - Capture Evaluation prior to full PCR using Phusion)[edit]

Background[edit]

    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[edit]

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

Results[edit]

  • Below is the gel image for the experiment:

File:Cw ZhangLab 2 2014-08-25 10hr 24min pp capture test phusion.jpg

  • Got some advice from Rui on things to do next:
    • Rui does notice that the NTC shows up in her experiments but usually sample has an overwhelmingly higher band. One observation she made was that perhaps the template concentration is lower than expected so sample is not much higher than NTC. Along that same line, the expected target band should be overwhelmingly brighter than the primer dimer band, which again might be related to the actual DNA template concentration used for the sample. I will test out the gDNA concentration again to double check what concentration I'm really adding
    • Also Rui noticed that the bands are slightly lower than expected (should be almost exactly between the 200bp-400bp band. Not quite sure why it is, so will need to think about that more