42.02 Cas gRNA complex formation

Procedure

1. Optionally, heat gRNA samples at 95ºC for 5 minutes and allow to slowly cool to room temperature for the correct secondary structure to form.
2. Prepare 10X NucleaSeq buffer:
   1. 49 uL 10 NucleaSeq buffer (no DTT)
   2. 1 uL 1M DTT
3. Combine a surplus of AsCas12a with gRNA (e.g., twice as much enzyme) in the 1X NucleaSeq buffer.
4. Incubate at 22C ("room temperature") for 30 min.
   • 10 min is likely more than enough.
5. Combine the following components:
   • Proteinase K concentration is likely independent of AsCas12a concentration as there is a huge surplus of Proteinase K.

Component	Vol. formula	Example (uL)
AsCas12a-gRNA complex	V	10
100 mM EDTA	V/10	1
Proteinase K	1	1

1. Incubate at 55C for 30 min.
   • You can also incubate at 37C for 30 min with no visible difference.
2. Heat inactivate samples at 95C 10 min.
3. To visualize:
   1. Keep samples on ice until loading.
   2. Run 5 uL of 10 ng (1 pmol) of sample
   3. Gel: 12% Native PAGE
   4. Pre-run: 10 min, 100 V
   5. Run: 60 min, 100 V
   6. Loading dye: 6X TriTrack, 1 uL
   7. Post-stain: SYBR Gold, 15 min