- Materials
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- 5 µg high molecular weight genomic DNA (recommended); 1–10 µg (or 0.1–2 pmol) can be used accordingly.
- Cas9 Sequencing Kit (SQK-CS9109)
- Flow Cell Priming Kit (EXP-FLP002)
- Consumables
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- S. pyogenes Cas9 Alt-R™ crRNAs (resuspended at 100 µM crRNA in TE pH 7.5)
- S. pyogenes Cas9 tracrRNA (e.g., IDT Alt-R™, Cat # 1072532, 1072533 or 1072534) resuspended at 100 µM in TE pH 7.5
- Alt-R® S. pyogenes HiFi Cas9 nuclease V3, 100 µg or 500 µg (IDT, Cat # 1081060 or # 1081061)
- Nuclease-Free Duplex Buffer (IDT Cat # 11-01-03-01)
- 1.5 ml Eppendorf DNA LoBind tubes
- 0.2 ml thin-walled PCR tubes
- Nuclease-free water (e.g. ThermoFisher, cat # AM9937)
- Agencourt AMPure XP beads (Beckman Coulter, A63881)
- Equipment
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- Microfuge
- Magnetic rack
- Vortex mixer
- Thermal cycler
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- P2 pipette and tips
- Ice bucket with ice
- Timer
- Optional equipment
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- Agilent Bioanalyzer (or equivalent)
- Qubit fluorometer (or equivalent for QC check)
- Eppendorf 5424 centrifuge (or equivalent)
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For this protocol, you will need 1–10 µg or 0.1–2 pmol high molecular weight genomic DNA (5 µg recommended).
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Cas9 Sequencing Kit contents (SQK-CS9109)
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Adapter Mix AMX Green 2 40 Ligation Buffer LNB Clear 2 200 Elution Buffer EB Black 1 200 Sequencing Buffer SQB Red 2 300 L Fragment Buffer LFB Orange 2 1,800 S Fragment Buffer SFB Grey 2 1,800 Loading Beads LB Pink 1 360 Phosphatase PHOS Brown tube, yellow label 1 50 TAQ Polymerase TAQ Brown tube, green label 1 15 SPRI Dilution Buffer SDB Brown tube, red label 1 1,200 T4 DNA Ligase LIG Brown tube, blue label 1 140 dATP dATP Brown tube, grey label 1 15 Reaction Buffer RB Brown tube, orange label 1 180 -
Flow Cell Priming Kit contents (EXP-FLP002)
Name Acronym Cap colour No. of vials Fill volume per vial (μl) Flush Buffer FB Blue 6 1,170 Flush Tether FLT Purple 1 200 -
Sourcing crRNA and tracrRNA
We recommend using synthetic crRNA and tracrRNA from IDT, which are of sufficient purity and carry modifications that confer stability and nuclease resistance. For this reason we caution against using single-guide RNAs (sgRNAs).
- S. pyogenes Cas9 Alt-R™ crRNAs
- S. pyogenes Cas9 tracrRNA (e.g. IDT Alt-R™, Cat # 1072532, 1072533 or 1072534)
Individual crRNAs and tracrRNA should be resuspended at 100 µM each in TE, pH 7.5, aliquoted to avoid freeze-thawing, and stored at –20° C for up to two weeks or –80° C if stored long-term. The crRNAs/tracrRNAs can be freeze-thawed a maximum of five times.
crRNAs may be pooled to make panels for generating multiple cuts in a single reaction. To pool crRNAs, we recommend dispensing equal volumes of each crRNA (up to 100 crRNAs, each at 100 µM) into a separate 1.5 ml Eppendorf DNA LoBind tube to make an equimolar crRNA mix.
We strongly recommend TE at pH 7.5, rather than pH 8.0, for the long-term stability of RNA oligos in storage.