- Materials
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- Ligation Buffer (LNB)
- Adapter Mix (AMX)
- T4 Ligase (LIG)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- Agencourt AMPure XP beads (Beckman Coulter™, A63881)
- Equipment
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- Ice bucket with wet ice
- Vortex mixer
- P1000 pipette and tips
- P100 pipette
- P20 pipette and tips
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Here, AMX adapters from the Cas Sequencing Kit (SQK-CS9109) are ligated to the ends generated by Cas9 cleavage.
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Thaw the Ligation Buffer (LNB) at room temperature, spin down and mix by pipetting. Due to viscosity, vortexing this buffer is ineffective. Place on ice immediately after thawing and mixing.
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Carefully transfer the contents of the 0.2 ml thin-walled PCR tube to a fresh 1.5 ml Eppendorf DNA LoBind Tube using a wide-bore pipette tip.
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Thaw an aliquot of Adapter Mix (AMX), mix by flicking the tube, pulse-spin to collect the liquid in the bottom of the vial, then return the vial to ice.
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Bring the AMPure XP beads to room temperature.
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Assemble the following at room temperature in a separate 1.5 ml Eppendorf DNA LoBind Tube, adding Adapter Mix (AMX) last, before you are ready to begin the ligation:
Reagent Volume Ligation Buffer (LNB) 20 µl Nuclease-free water 3 µl T4 Ligase (LIG) 10 µl Adapter Mix (AMX)* 5 µl Total 38 µl -
Mix by pipetting the above ligation mix thoroughly. Ligation Buffer (LNB) is very viscous, so the adapter ligation mix needs to be well-mixed.
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IMPORTANT: Add 20 µl of the adapter ligation mix to the cleaved and dA-tailed sample. Mix gently by flicking the tube. Do not centrifuge the sample at this stage. Immediately after mixing, add the remainder of the adapter ligation mix to the cleaved and dA-tailed sample, to yield an 80 µl ligation mix.
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Ensure the components are thoroughly mixed by pipetting, and spin down.
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Incubate the reaction for 10 minutes at room temperature.