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== Test different dNTPs and Phusion U for capture == * I want to be able to capture using less input DNA * Testing 63 ng of input, with more polymerase and adjusting the dNTP concentrations. * Prepare oligo mix: 200:1 ratio for 360 ng. * Capture mixture: Oligo mix: 52.03 uL 10x Ampligase buffer: 8 uL ------------------------- Total: 60.03 uL * Aliquot: 52.49 uL for samples + 4.47 uL gDNA (315 ng of biscvt GM12878) + 13.04 uL water = 70 uL master mix. * Aliquot: 7.54 uL (1/8) for NTC + 2.46 uL water = 10 uL NTC reaction. * Split the 70 uL master mix into 5 tubes (14 uL, ~63 ng in each). * Calculate dNTP required: 63 ng/ 3 pg = 21x10^3 copies 460,000 probes x 21E10^3 x 300 nt = 4.81 pmoles dNTPs x 10 = 48.1 pmoles dNTPs * Will be adding 2 uL of PLN mixture: 25 uM dNTPs ( to add a total 50 pmoles to each reaction) 0.35 U/uL ligase ( to add 1U/20 uL ) 1.75 U/uL polymerase ( to add 5U/20 uL ) * Conditions for 5 reactions: #1 - HK + PU, 25 uM dNTPs #2 - HK + PU, 100 uM dNTPs #3 - HK, 25 uM dNTPs #4 - PU, 25 uM dNTPs #5 - HK, 100 uM dNTPs *
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