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=== Capture set up === ==== Things to do in order ==== * Set up capture reaction in total 25 μL in 8-tube strip (96-well plate if more samples are processed) with the following components: 400 ng of WGBS DNA, 200:1 of probe to target ratio and 1x Ampligase Buffer. # Calculate volume of WGBS libraries required for 400ng ## Adjust total volume to 50ul (could be any number) ## Evaporate WGBS libraries in 1.5mL LoBind tube to reduce total volume to fit 25ul capture reaction including probes and 10X Ampligase Buffer ## Resuspend evaporated WGBS libraries with H2O and transfer to 0.2mL 8-tube strip or 96-well plate based on number of sample # Calculate the amount of each probe set required. The amount is based on probe to target ratio and template amount. # Prepare probes and 10X Ampligase buffer mix ## Add probe/buffer mix to the tube with WGBS libraries ## Mix well (spinand layer with equal volume of mineral oil (25ul of mineral oil to 25ul of capture reaction) ## Spin down the tube again before incubate on thermocycler as the following program: 94 ºC for 30 s, gradually lower temperature to 55 ºC at 0.02 ºC/s in a thermocycler, and let hybridize at 55 ºC for 20 hours (This is requirement. Not recommend to incubate shorter than 20h) ==== WGBS libraries ==== * We require 400ng of WGBS library for BSPP capture. The total volume of 400ng is quite large and exceed 25ul which is the total volume of BSPP capture. I simply pipette 400ng of WGBS libraries to 1.5mL LoBind tube. Then add H2O to bring total volume to 50ul. {| {{table}} border = 1 | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''Conc. (ng/ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Amount in 70ul (ng)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume for 400ul (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Sample''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O to 50ul''' |- | KU_1||9.89||692.30||40.44||KU_1||9.56 |- | KU_2||14.90||1,043.00||26.85||KU_2||23.15 |- | KU_3||11.00||770.00||36.36||KU_3||13.64 |- | KU_4||14.30||1,001.00||27.97||KU_4||22.03 |- | KU_5||10.10||707.00||39.60||KU_5||10.40 |- | KU_6||12.70||889.00||31.50||KU_6||18.50 |- | KU_7||15.60||1,092.00||25.64||KU_7||24.36 |- | KU_8||9.22||645.40||43.38||KU_8||6.62 |- | KU_9||9.09||636.30||44.00||KU_9||6.00 |- | KU_10||8.17||571.90||48.96||KU_10||1.04 |} * I evaporated WGBS libraries by closing the lid of the tube and made a hole by poking with needle size G22 (3 hole/tube) and evaporate at 60C for ~15min. The left over volume was between 5-12ul. I think to make it simple, I just dry up the whole tube then resuspend with equal volume of H2O to fit the capture volume. In this experiment I adjust total volume of WGBS library to 12ul with H2O ==== Probe Buffer Mix ==== {| {{table}} border = 1 | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''11.5 rxn mix''' |- | MONOD V5A (LMS) (12.1 ng/ul)||8.93||102.70 |- | CRC (13.2ng/ul)||0.28||3.22 |- | 10X Ampligase Buffer||2.50||28.75 |- | H2O||1.29||14.84 |- | Total||13.00|| |} '''Program''' -> 95c 5min -> cool down to 55C at 0.02C/sec -> 55C 20h -> add 2.5ul PLN mix (20%v/v Hemo Klentaq; 0.5U/ul AmpLigase; 100uM dNTP) --> '''No need to change program while adding PLN mix (keep the tube at 55C all the time)''' -> 55C 20h-> 94C 2min -> add 2ul Exo I/III mix-> 37C 2h -> 94C 5min -> 10C hold. --> '''Press proceed once''' before adding ExoI/III and waiting until temperature down to 37C before adding ExoI/III ==== Actual thermocycle program ==== :- 1: 95C 5min :- 2: 94.8C, 30s ::- -0,2C/cycle (increment -0.2C/cycles) :- 3: Go to 2 193 times :- 4: 55C forever :- 5: 94C, 2min :- 6: 37C, 2h :- 7: 94C, 5min :- 8: Hold at 4-10C forever or continue to PCR * Note: After adding PLN, the incubation time could be varied between 2-20h.
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