Chris:LabNotes/FateMapping/Calendar/2014/2014-9-3
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Target capture - Get Rui's probes (positive control) working (Part II)[edit]
Background[edit]
- This is continuation of the procedure from <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-9-2>
- As a reminder, below is a description of the reactions/conditions were are doing for this experiment:
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]
- We are going to use the previous target captured circularlized DNA as template for Phusion reaction (Phusion was newly ordered for this experiment and can be found in my enzyme box in the -20C fridge)
- 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) 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