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== Test capture subset A == === Capture setup === {| 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 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''' |- | GM-A-1-55||GM12878||150.0||2.400||6.00||2.00||9.60||20.00 |- | GM-A-5-56.4||GM12878||150.0||2.400||6.00||2.00||9.60||20.00 |- | GM-A-8-58.8||GM12878||150.0||2.400||6.00||2.00||9.60||20.00 |- | GM-A-12-60||GM12878||150.0||2.400||6.00||2.00||9.60||20.00 |} === Capture program === 1. 95°C 30s 2. 94.8°C 30s -0.2°C per 30s 174 times -> 60°C 3. Gradient: 55/56.4/58.8/60 °C 20h ADD 2.5 uL SLN mix 4. Gradient: 55/56.4/58.8/60 °C 20h 5. 94°C 2m ADD 2.0 uL ExoI/ExoIII mix 6. 37°C 2h 7. 90°C 5m 8. 4°C forever === Capture PCR === * 4 uL of capture was used to amplify. Two step PCR with 8 cycles first step, and 10 cycles second step = 18 cycles of PCR total for all samples. * TBE Gel: [[File:HOTSPOTS_captureA_test.jpg | 300px]] [[File:HOTSPOTS_captureA_test_repeat.job | 300px]] Gel might be slightly shifted or 471 bp calculation is wrong? The expected band seems to be greater than 500bp. The best annealing temperature appears to be 56.4 degC. This temperature should apply to all the subsets since the probes were randomly assigned to subsets. * Will try capture subset B, C, D, E, and F at 56.4 degC. Also, I will amplify 19 or 20 cycles to hopefully increase yield of capture amplicons.
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