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]
- Continued from <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-8-18>
- Relatively confident that we should be using Phusion to avoid strand displacement, which seems to confound sequencing results when using KAPA
- Have 13 rxn's (testing different probe:target ratios along with inclusion of more controls such as multiple negative controls on every step and the inclusion of Rui's probes)
- Below is the experimental design again:
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]
- 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) 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