- Materials
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- 5 µg high molecular weight dephosphorylated genomic DNA (recommended); 1 - 10 µg (or 0.1-2 pmol) can be used accordingly.
- crRNA-tracrRNA-Cas9 ribonucleoprotein complexes (RNPs)
- Taq Polymerase (TAQ)
- dATP (dATP)
- Consumables
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- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Equipment
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- Thermal cycler
- Ice bucket with wet ice
- Vortex mixer
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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In this step, Cas9 RNPs (see 'Preparing the Cas9 ribonucleoprotein complexes') and Taq polymerase are added to the dephosphorylated genomic DNA sample.
This process cleaves target and dA-tails all available DNA ends in one step, activating the Cas9 cut sites for ligation.
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Thaw the dATP tube, vortex to mix thoroughly and place on ice.
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Spin down and place the tube of Taq Polymerase (TAQ) on ice.
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To the PCR tube containing 30 µl dephosphorylated DNA sample, add:
Reagent Volume Dephosphorylated genomic DNA sample 30 µl Cas9 RNPs 10 µl dATP 1 µl Taq Polymerase (TAQ) 1 µl Total 42 µl -
Carefully mix the contents of the tube by gentle inversion, then spin down and place the tube in the thermal cycler.
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Using the thermal cycler, incubate at 37ºC for 15-60 minutes*, then 72ºC for 5 minutes and hold at 4ºC or return to tube to ice.