Chris:LabNotes/FateMapping/Calendar/2014/2014-9-3

From ZhangLabWiki
Jump to navigation Jump to search

Target capture - Get Rui's probes (positive control) working (Part II)[edit]

Background[edit]

    rxn     Target (ul)     Probe (ul)     Water (ul) -> to get to 18ul total volume
    1A      10              3.30 (CW)      4.70** => Incubate for 5min 95C for initial denaturing
    1B      10              3.30 (CW)      4.70 => Put in after 4.5min 95C to have a total of 30sec denaturing
    2A      10              1.60 (RL)      6.40**
    2B      10              1.60 (RL)      6.40
    3A       0              3.30 (CW)      14.70**
    3B       0              3.30 (CW)      14.70
    4A       0              1.60 (RL)      16.40**
    4B       0              1.60 (RL)      16.40
    5A       0              0              20.00**
    5B       0              0              20.00

Procedure[edit]

Component                     1x(ul)    14x(ul)      Init Conc     Final Conc
H2O                          2.6         36.4        --            --
2x Phusion Flash PCR MM       10          140        2x            1x
50x Sybr Green                .4          5.6        50x           1x 
AmpF6.4Sol                     1           14        10uM          .5uM
AmpR6.3.Indx (Indx40)          1           14        10uM          .5uM
Circularized DNA Template      5          --         --            --
                              20
  • Also will include 2 PCR NTC's (rxn 6 for each 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 (used 12 cycles in the end)
72C 3min
Hold 15C

Results[edit]

  • Below is the gel image for Strip A (5min 95C initial denaturing)

File:Cw ZhangLab 2 2014-09-03 20hr 58min pp capture phusion strip A.jpg

  • Below is the gel image for Strip B (30sec 95C initial denaturing)

File:Cw ZhangLab 2 2014-09-03 21hr 00min pp capture phusion strip B.jpg