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== Adapter ligation == * Adapter ligation mix (Total 50ul) : Ligation reaction mix 49.2ul/reaction & 0.8ul of 3uM adapters (Diluted 30uM adapters 10X with cold 1X Stoffel buffer) === Adapter preparation === * Prepare 30uM adapters {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''10ul, 30uM''' |- | 10XStoffel buffer||1.00 |- | 100uM PE_t_adapter||3.00 |- | 100uM PE_b_adapter||3.00 |- | H2O||3.00 |} * 94C 2min -> 0.2C/sec to 20C -> 4C hold * Dilute 10X by mixing 3ul of 30uM adapters with 27ul 1X Stoffel buffer (keep annealed adapters and buffer on cold box all the time) * Store 30uM annealed adapter at -20C for future use === Ligation reaction mix === {| class="wikitable" style="text-align:center; | align="center" style="background:#f0f0f0;"|'''Ligation reaction mix''' | align="center" style="background:#f0f0f0;"|'''1x rxn''' | align="center" style="background:#f0f0f0;"|'''13x rxn mix''' |- | KAPA 5X Ligation buffer||10.00||130.00 |- | KAPA DNA ligase||5.00||65.00 |- | H2O||20.20||262.60 |- | Total||35.20||457.60 |} * Aliquot 35.2 ul of ligation reaction mix to each tube (I prefer using a clear tube since I can see from the side during bead purification) * Transfer 4ul of sample to reaction tubes ** If the sample is already in a clear tube, just add ligation reaction mix directly to fragmented DNA and adjust volume in ligation reaction mix ** I used 0.2 ml PCR Tube Strips, low-Profile tube (Biorad) to set up reaction * Rinse the tube with 10ul H2O and transfer all to reaction tube (total volume now is 49.2ul) * Note: for NTC, I accidentally added too much H2O, so total of reaction of NTC was 60.8ul * Add 0.8ul of 3uM adapters * 20C for 15min (no shaking) * Bead purification: 50ul AmPure beads mix by pipetting 10 times, wait 10min (no shaking), sit on magnet 5min, wash twice with fresh 80% EtOH, dry for 3 min ** I was with 160ul of 80% EtOH since I used low profile tube. * Eluted with 15ul H2O (used all for PCR)
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