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== Test capture - 4 reactions == === Pool subset probes for 4 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;"|'''Amount for 4.5 capture (ng)''' | align="center" style="background:#f0f0f0;"|'''Subset Size''' |- | A||100||81.2||8.12||12.88||104.55||90000 |- | B||100||46.4||4.64||22.53||104.55||90000 |- | C||100||84.8||8.48||12.33||104.55||90000 |- | D||100||113||11.3||9.25||104.55||90000 |- | E||100||84.7||8.47||12.34||104.55||90000 |- | F||100||97.4||9.74||0.78||7.61||6553 |} * Pool probes for 4.5 captures and aliquot 15.58 uL per capture. === Capture setup=== * I used up all the bisulfite converted gDNA because the converted DNA cannot be kept for long period of times. {| 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''' |- | 1. 54 Ctrl||H1 p54 Control TeSR||193.90||7.00||16.00||3.00||4.00||30 |- | 2. Act||H1 p54 Activin-A||362.00||10.00||16.00||3.00||1.00||30 |- | 3. 47 Ctrl||H1 p47 Control TeSR||144.95||6.50||16.00||3.00||4.50||30 |- | 4. Bmp||H1 p47 BMP-4||205.60||8.00||16.00||3.00||3.00||30 |} === Capture program === 1. 95°C 30s 2. 94.8°C 30s -0.2°C per 30s 198 times 3. 55°C 20h ADD 2.5 uL SLN mix 4. 55°C 19.5h 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 === * Amplified 4 uL in 25 uL reaction using KAPA 2xMM with AmpR6.3Ind17 REAGENT 1x 5.5x Capture template 4.00 uL -- 10 uM AmpF6.4Sol 0.50 uL 2.75 uL 10 uM AmpF6.3RInd17 0.50 uL 2.75 uL 2xKAPA Master Mix 12.50 uL 68.75 uL Nuclease free H2O 7.50 uL 41.25 uL * TBE Gel: [[File:HOTSPOTS_capture1_test.jpg | 300px]]
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