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)
Background
- 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