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]
- 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[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) 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