- Consumables
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- S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
- Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
- S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
- Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
- 0.2 ml thin-walled PCR tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Equipment
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- Thermal cycler
- Ice bucket with wet ice
- P200 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
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Pre-heat a thermal cycler to 95ºC.
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Thaw an aliquot of Reaction Buffer (RB), mix by vortexing, and place on ice.
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In an 1.5 ml Eppendorf DNA LoBind tube, pool the crRNA probes for each cleavage reaction by combining equal volumes of each crRNA probe, resuspended at 100 µM in TE (pH 7.5).
- A single crRNA or many crRNA probes (up to ~100) may be used in a single cleavage reaction.
- The crRNA probes may also be pre-mixed as an off-catalogue request from IDT.
- For example, probes for the HTT gene, found here, can be used as an individual experiment or in addition to other probes as an in-run control.
- Unused crRNA probe mix may be stored at -80ºC and minimal freeze thaw recommended.
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Anneal the pooled crRNAs with tracrRNA in Duplex Buffer by assembling the following in a 0.2 ml thin-walled PCR tube, as follows:
Reagent Volume Duplex buffer 8 µl crRNA pool (100 µM, equimolar) 1 µl tracrRNA (100 µM) 1 µl Total 10 µl -
Mix well by pipetting and spin down.
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Using a thermal cycler heat the above reaction mix at 95ºC for 5 mins, then remove the tube from the thermal cycler and allow it to cool to room temperature, then spin down the tube to collect any liquid in the bottom of the tube.
- Storage and reuse of the annealed mix is not recommended.
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To form Cas9 RNPs, assemble the components in the table in an 1.5 ml Eppendorf DNA LoBind tube; this will form the annealed crRNA•tracrRNA, through pooling in the stated order:
Reagent Volume Nuclease-free water 79.2 µl Reaction Buffer (RB) 10 µl Annealed crRNA•tracrRNA pool (10 µM) 10 µl (Step 4, above) HiFi Cas9 (62 µM) 0.8 µl Total 100 µl Note: Refer to the tip below for scaling-down this ternary RNP mix.
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Mix thoroughly by flicking the tube.
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Form the RNPs by incubating the tube at room temperature for 30 minutes, then return the RNPs on ice until required.