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=Target capture with padlock probes (designed 5/12) test (Part III - Capture Evaluation prior to full PCR using Phusion)= ==Background== *Continued from <http://genome-tech.ucsd.edu/LabNotes/index.php/Chris:LabNotes/FateMapping/Calendar/2014/2014-6-20> *After talking with Alice and Dinh, there are a few possibilities to try next: **Alice and Dr. Zhang suggested to increase the annealing temperature for probe capture, since the GC content for the gDNA is higher than for bisulfite converted DNA. Will attempt capture again but with a gradient PCR method maybe from 55 to 64 (check the recommended Tm/annealing temperature of the probes designed). **Dinh suggested to use Phusion instead of KAPA because Phusion will not have any strand displacement activity. Also, Dr. Zhang suggested to ask Din about how to check whether they target unique genomic regions. In order to do this, will need to do sequencing and look at the UMI's that were added to each probe then map. *Today's plan will be to test out Phusion and see if that helps with getting rid of a few of the confounding bands in the gel. ==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) 10x(ul) Init Conc Final Conc H2O 2.6 26 -- -- 2x Phusion Flash PCR MM 10 100 2x 1x 50x Sybr Green .4 4 50x 1x AmpF6.4Sol 1 10 10uM .5uM AmpR6.3.Indx (Indx39) 1 10 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== *Below is the gel image of the PCR: [[File:cw_ZhangLab_2_2014-06-20_18hr_13min_capture_pcr_test_phusion_enzyme.jpg|500px]] *Below is a table describing each lane and the amount of template for each Rxn # Probe:Target Lane Amount Added (ul) 1 250:1 1 5 2 250:1 2 5 3 500:1 3 5 4 500:1 4 5 5 1000:1 5 5 6 1000:1 6 5 7 250:1 (NTC) 7 5 8 500:1 (NTC) 8 5 9 1000:1 (NTC) 9 5 10 PCR NTC 10 5 -- Low Mass 11 1 -- 100bp 12 2
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