- Materials
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- Adapter Mix (AMX)
- Consumables
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- NEB Blunt/TA Ligase Master Mix (NEB, cat # M0367)
- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- Equipment
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- Microfuge
- Ice bucket with ice
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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Check the contents of each tube are clear of any precipitate and are thoroughly mixed before setting up the reaction.
- Mix the contents of each tube by flicking
- Check that there is no precipitate present (DTT in the Blunt/TA Master Mix can sometimes form a precipitate)
- Spin down briefly before accurately pipetting the contents in the reaction
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Taking the end-prepped DNA, perform adapter ligation as follows, mixing by flicking the tube between each sequential addition.
Adapter ligation and tethering demo
Reagent Volume End-prepped DNA 30 µl Adapter Mix 5 µl Blunt/TA Ligation Master Mix 50 µl Nuclease-free water 15 µl Total 100 µl -
Mix gently by flicking the tube, and spin down.
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Incubate the reaction for 10 minutes at room temperature.