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== End repair & dA-tailing == *Note: I will reduce the reaction volume to be half of the instruction and reduce the reagent volume/amount proportionally. * KAPA Hyper Prep reaction set up (exactly in the instruction): :- 50ul of fragmented DNA :- 7ul of End repair & dA-tailing buffer :- 3ul of End repair & dA-tailing enzyme mix :- Total reaction: 60ul ==== End repair & dA-tailing reaction mix ==== {| {{table}} border = 1 | align="center" style="width:240px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''half rxn''' | align="center" style="width:80px;background:#f0f0f0;"|'''11.2x rxn''' |- | End repair/dA-tailing buffer ||7.00||3.50||39.20 |- | End repair/dA-tailing enzyme mix||3.00||1.50||16.80 |- | H2O||6.00||3.00|| |- | Total||10.00||8.00|| |}<br> ==== Reaction set up ==== {| {{table}} border = 1 | align="center" style="width:140px;background:#f0f0f0;"|'''Sample IDs''' | align="center" style="width:80px;background:#f0f0f0;"|'''Zhang lab ID''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume''' | align="center" style="width:80px;background:#f0f0f0;"|'''End repair/dA-tailing rxn mix''' | align="center" style="width:80px;background:#f0f0f0;"|'''H2O (ul)''' | align="center" style="width:80px;background:#f0f0f0;"|'''Total (ul)''' |- | 9.H3.25||KU_1||22.00||8||0.00||30.00 |- | 9.I19.36||KU_2||22.00||8||0.00||30.00 |- | 9.F20.56||KU_3||22.00||8||0.00||30.00 |- | 9.H5.58||KU_4||22.00||8||0.00||30.00 |- | 9.G9.25||KU_5||22.00||8||0.00||30.00 |- | 9.I7.3||KU_6||22.00||8||0.00||30.00 |- | 9.H7.69||KU_7||22.00||8||0.00||30.00 |- | 9.C28.47||KU_8||22.00||8||0.00||30.00 |- | 9.B7.36||KU_9||22.00||8||0.00||30.00 |- | 10.A13.3||KU_10||22.00||8||0.00||30.00 |- | NTC||NTC||0.00||8||25.00||33.00 |} :- Add 8ul of End repair/dA-tailing rxn mix to each tube in 8-tube strip :- Transfer 22ul of ctDNA to each rxn tube. :- Mix well (spin down & pulse-vortexing 10X & spin down) :- 20C, 30min :- 65C, 30min, :- Hold 4C :- Proceed to Adaptor ligation immediately
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