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== Adaptor ligation == ''' Reaction set up''' {| {{table}} | align="center" style="background:#f0f0f0;"|'''Components''' | align="center" style="background:#f0f0f0;"|'''Volume (ul)''' |- | Fragmented DNA||6.00 |- | 1.5uM NEBNext adaptor||1.67 |- | Ligation reaction mix||42.33 |- | Total||50.00 |} ''' NEBNext adaptor preparation ''' * I diluted a new batch of adaptor by diluting 15uM adaptor to 1.5uM (mix 20ul of 15uM NEBNext adaptor with 180ul H2O) and aliquoted 25ul into 8-striptubes and saved at -20C after used. ''' 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;"|'''24x rxn mix''' |- | 2X KAPA Rapid Ligation Buffer||25.00||600.00 |- | KAPA Rapid T4 DNA Ligase||2.00||48.00 |- | H2O||15.33||367.92 |- | Total||42.33||1,015.92 |} * Aliquot 42.33 ul of ligation reaction mix to each tube containing 6ul of fragmented DNA * Add 1.67ul of 1.5uM NebNext loob adaptor. Mix well by pipetting 15-20 times and quick spin. * 25C for 10min * Add 2ul USER. Mix well by pipetting 15-20 times and quick spin. 37C for 15min ** For the library prep using NEBNext® DNA Library and this loop adaptor, they used 3ul USER in total volume 50ul or 83.5ul, 37 C for 15 min. I used a little less '''Bead purification''' * 50ul AmPure beads mix by pipetting 10 times * Wait 10min * Sit on magnetic stand 5min * Wash twice with freshly prepared 80% EtOH (after adding 80% EtOH, I wait for 30sec before pipette up and down few times and discard spnt) ** I wash with 160ul of 80% EtOH since I used low profile tube. * Dry the beads for 3 min * Eluted with 20ul H2O (resuspend and wait for 2min, used all for PCR)
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