Cas9 Condition Optimization

This protocol runs several in vitro Cas9 cleavage conditions for identifying the optimal setup for a specific target (in this case the Sub1 gene).

Cas9 Cleavage

  1. Cas9 nuclease reactions can be set up as described in the table below. For identifying cleavage conditions, use a combination of 1X and 2X Cas9 reactions along with no sgRNA controls. See Table 1.

  2. Incubate the reaction mix for 10 minutes at 37 C.

  3. Add 4 ul of DNA substrate.

  4. Incubate the reaction for 2 hours at 37 C.

  5. Denature the Cas9 nuclease with a 10 minute incubation at 80 C. If necessary, perform a chloroform extraction on the digestion products.

Chloroform Extraction

  1. Aliquot 1 ml chloroform to use as a stock solution.

  2. Add 1 volume of chloroform to the microfuge tube containing Cas9 reaction solution. Gently pipette up and down 10 times.

  3. Centrifuge the tube and aliquot the top layer to a new microfuge tube. This solution may be used for electrophoresis.

  4. Leave the tube of stock solution chloroform in the hood until the chloroform evaporates. 

MetaPhor Agarose Gel Electrophoresis

  1. Prepare a 3% agarose gel with the following components in a 1 L flask:
  • 5 g MetaPhor agarose
  • 10 g regular agarose
  • 50 ml 10X TAE buffer
  • ddH2O to 500 ml. Mark a line on the flask at the 500 ml volume.
  1. Autoclave the flask for 20-30 minutes. 

  2. Pour the solution into autoclaved flasks or bottles in 100 ml volumes when the solution cools slightly. If the volume is below the marked 500 ml line, add additional TAE buffer before pouring.

  3. To cast the gel, gradually microwave a flask or bottle with 100 ml of the agarose mix. Heat the mix until it is melted but cool enough to pour.

  4. Pour the mix into the gel molds. It takes approximately 40-45 ml for one gel. 

  5. Prepare a normal 3% agarose gel as well with 1.5 g agarose in 50 ml TAE buffer.

  6. Prepare the 20 ul samples with 4 ul 6X loading dye.

  7. Add 12 ul of prepared sample to each gel as indicated in table 2.

  8. Run the gel until the bands have migrated at least ⅔ of the gel length. 

  9. Stain the gel with EtBr and visualize it with UV.

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