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Cas-gRNA complex formation

Duration: 1 h 40 min + 1h 20 min for gel = 3 h

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