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== Adaptor ligation == ==== Adaptor ligation reaction mix ==== * I prepare the new tube of 1:20 diluted TruSeq adaptor (conc. after dilution = 714nM) Index3. Since Dr. Zhang does not want to sequence the library, I just used the same barcoded adaptor for all samples. * I also diluted a customized Y-adaptore 1:20 with 1X Stoffel buffer to be easier for ligation reaction mix prep. * I prepared two tube of ligation reaction mix containing TruSeq adaptor and customized adaptore with UMI {| {{table}} border = 1 | align="center" style="width:200px;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;"|'''4.1x rxn''' |- | Ligation buffer||30.00||15.00||61.50 |- | DNA ligase||10.00||5.00||20.50 |- | H2O||8.00||4.00||16.40 |- | Total||48.00||24.00||98.40 |- | 1:20 diluted adaptor (714nM)||2.00||1.00||4.10 |- | Total||50.00||25.00|| |} * Final conc. of adaptor in ligation reaction is 13.64nM (the kit recommends 14nM) ==== Adaptor ligation reaction set up ==== {| {{table}} border = 1 | align="center" style="width:200px;background:#f0f0f0;"|'''Components''' | align="center" style="width:80px;background:#f0f0f0;"|'''Volume (ul)''' |- | End repair & dA-tailing rxn product||30.00 |- | Adaptor ligation rxn mix||25.00 |- | Total||55.00 |} * For a full reaction, I add reagent directly to the tube woth 2ul of 1:20 diluted adaptor :- Add 25ul of adaptor ligation reaction mix :- Mix well :- 20C, 15min :- Continue to SPRI clean up immediately ==== <u>1X AMPure bead purification</u> ==== * The instruction suggested to use 0.8X of AMPure bead for clean up, but I use 1X of AMPure bead - Aliquot AMPure bead in 1.5mL tube and leave at RT :- Add 55ul AMPure bead for a half reaction and 110ul for a full reaction. Mix by pipetting 10x :- Sit for 15min :- Transfer to sit on magnet for 5min :- Wash twice with 200ul freshly prepared 80% EtOH :- Dry the bead for 3-5min :- Resuspend with 25ul EB buffer
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