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== Test capture 56-57°C, 300 ng template, subset C only == * Noi performed bisulfite conversion for GM12878, concentration by Qubit is 31.1ng/uL. * I used subset C here, which had the poorest efficiency from capture at 56.4 degC. I repeat the capture here for C because I did not expect subset C to perform worse than A,B,D, and E because the probes were randomly assigned to subsets. At least if I can find the proper annealing temperature for C, then it should also work fine for A,B,D, and E. ===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''' |- | N-GM-C-3-56.1||GM12878||300.0||9.65||5.48||2.00||2.87||20.00 |- | N-GM-C-6-56.4||GM12878||300.0||9.65||5.48||2.00||2.87||20.00 |- | N-GM-C-9-56.9||GM12878||300.0||9.65||5.48||2.00||2.87||20.00 |} ===Capture program=== 1. 95°C 30s 2. 94.8°C 30s -0.2°C per 30s 174 times -> 60°C 3. Gradient: 56.1/56.4/56.9 °C 20h ADD 2.5 uL SLN mix 4. Gradient: 56.1/56.4/56.9 °C 20h 5. 94°C 2m ADD 2.0 uL ExoI/ExoIII mix, add 10 uL water 6. 37°C 2h 7. 90°C 5m 8. 4°C forever ===Capture PCR=== * Amplified 4 uL for 21 (8 + 13) cycles as test PCR. * qPCR plot [[File:HOTSPOTS_56.1to56.9capture.bmp | 600px]] * gel image: loaded 10 uL in first 3 lanes, and 5 uL in last 3 lanes ** I always get multiple bands. The top & clearest band is between 500-600bp. The expected band should be 471 bp, so that should be within the smear in between 400 and 500 bp. ** Since I did not run this gel for long enough, I could not see a clear separation for the 471 bp band. ** Capture at 56.1 degC produce the topmost band, whereas before with 55 degC, I was not able to see bands larger than 400bp. 56.4 degC appears to be the best annealing temperature. [[File:HOTSPOTS_56.1to56.9capture_PCR.jpg | 300px]] * I might split capture product in two, each half will be amplified with a different barcode & perform gel cut for (1) the topmost band between 500-600bp, (2) the middle band between 400-500bp. The two libraries can be spiked in with Noi's MONOD samples for sequencing. Since capture with 56.4 and 56.9 degC are similar, I will pool them together to increase the amount of product. * Pool 56.4 and 56.9 degC reactions together and split to perform PCR (22 cycles) with Index Primers 28 and 29. 1x 7x Capture products 4.0 uL 28.0 uL 10 uM forward 0.5 uL 3.5 uL 10 uM reverse(28,29) 0.5 uL 3.5 uL KAPA 2X MM 12.5 uL 87.5 uL Water 7.5 uL 52.5 uL TOTAL 25.0 uL 175.0 uL * Size selection [[File:HOTSPOTS_Indx28,Indx29_sizeselect.jpg | 300px]]
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