Cas12a (Cpf1) Cleavage Reaction

This is for a standard 30 µL reaction. Vary as you wish. The important things are that the RNA:Cas12a ratio be 2:1, and that you take into account the salt in the Cpf1 buffer (since we gel filter with high salt it’s actually quite significant).

Contents

1. Buffer
2. Procedure

1. Buffer

• 100 mM NaCl (final - take into account the salt in the Cas12a tube)
• 50 mM Tris HCl pH 8.0
• 5 mM MgCl₂
• 2 mM DTT

2. Procedure

1. In a PCR tube, add in this order:
   1. Buffer (29 µL less the volume of RNA and protein you’re about to add).
   2. RNA (100 nM final).
   3. Cas12a (50 nM final).
2. Make three control tubes: one without RNA, and one without protein, and one without RNA or protein.
3. Vortex and spin down the tubes. There is a PCR tube microfuge in the radioactive room.
4. Incubate in a PCR machine at 25°C for 15 minutes.
5. Spike in 1 µL of 150 nM DNA to each tube. Vortex briefly and spin down.
6. Incubate in a PCR machine at 25°C for 60 minutes.
7. Add 1 µL of 300 mM EDTA to each tube to quench the reaction, and 1 µL Proteinase K.
8. Incubate at 52°C for 20 minutes.
9. Run samples on an agarose gel, with nicked and linearized plasmids.