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== Test capture using full probes set == * Perform capture using 460K probes in one reaction. * Amount sample will be 300 ng and annealing temperature will be set at 56.4 degC. * Vary probes to target ratio: 100:1, 50:1, 30:1 === Prepare mixed probes=== * Mix probes for 100:1 ratio, 300 ng template, for 1.85 reactions. {| class="wikitable" cellpadding=1 | align="center" style="background:#f0f0f0;"|'''Subset''' | align="center" style="background:#f0f0f0;"|'''Dilution''' | align="center" style="background:#f0f0f0;"|'''QUBIT(ng/mL)''' | align="center" style="background:#f0f0f0;"|'''Conc(ng/uL)''' | align="center" style="background:#f0f0f0;"|'''Volume(uL)''' | align="center" style="background:#f0f0f0;"|'''Mass(ng)''' | align="center" style="background:#f0f0f0;"|'''Subset Size''' |- | A||100||107||10.7||8.03||85.96||90000 |- | B||100||71.2||7.12||12.07||85.96||90000 |- | C||100||86.1||8.61||9.98||85.96||90000 |- | D||100||150||15||5.73||85.96||90000 |- | E||100||85.1||8.51||10.10||85.96||90000 |- | F||100||53.5||5.35||1.17||6.26||6553 |} === Capture setup === * Aliquot probes & concentrate before adding buffer and other reagents. {| class="wikitable" cellpadding=1 | align="center" style="background:#f0f0f0;"|'''Tube ID''' | align="center" style="background:#f0f0f0;"|'''Converted sample''' | align="center" style="background:#f0f0f0;"|'''Amount sample (ng)''' | align="center" style="background:#f0f0f0;"|'''Volume sample (uL)''' | align="center" style="background:#f0f0f0;"|'''Volume mixed probes (uL)''' | align="center" style="background:#f0f0f0;"|'''Volume of 10x ampligase buffer''' | align="center" style="background:#f0f0f0;"|'''Volume water (uL)''' | align="center" style="background:#f0f0f0;"|'''Total volume''' |- | N-GM-100||GM12878||300.0||9.65||25.46||2.00||-17.11||20.00 |- | N-GM-50||GM12878||300.0||9.65||12.73||2.00||-4.38||20.00 |- | N-GM-30||GM12878||300.0||9.65||7.64||2.00||0.71||20.00 |} === Capture PCR === * Amplify 28 uL in 175 uL (split into 2 tubes) Indx33 100:1 Indx35 50:1 Indx37 30:1 * qPCR plot [[File:HOTSPOTS_GM12878_capture_probeRatios.bmp | 600px]] * TBE gel [[File:HOTSPOTS_GM12878_capture_probeRatios.jpg | 300px]]
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