Protocols and instructions

Micro-injection ready reagent preparation (No Donor):

Recommended storage: -80℃

Preparation instructions:
  • Thaw 1 aliquot* of premixed micro-injection ready CRISPR-Cas9 reagents and place on ice immediately.
  • Spin the sample down in the MI ready vial at 10,000rpm (or top speed) for 10 minutes at 4℃. Transfer the top 25µL-30µL of supernatant into a new microtube and place on ice immediately.
  • The reagent is ready to be micro-injected.

*It is recommended to not freeze/thaw the reagents more than twice.

 

Micro-injection ready reagent paired with donor preparation:

Recommended storage: -80℃

Preparation instructions:
  • Thaw 1 aliquot of premixed micro-injection ready CRISPR-Cas9 vial and 1 aliquot of donor vial and place on ice immediately.
  • Pipet 50µL from each vial of micro-injection ready CRISPR-Cas9 and Donor and mix 1:1 in a new microtube.
  • Spin the sample down in the MI ready vial at 10,000rpm (or top speed) for 10 minutes at 4℃. Transfer the top 25µL-30µL of supernatant into a new microtube and place on ice immediately.
  • The reagent is ready to be micro-injected.

*It is recommended to not freeze/thaw the reagents more than twice.

 

Standard Reagent used for micro-injection preparation:

Recommended storage: -80℃

Preparation instructions:
  • Thaw each vial of Cas9 (mRNA or RNP), sgRNA, and donor if applicable and immediately place on ice.
  • Make appropriate dilutions (if necessary) to obtain desired final micro-injection concentrations using provided buffer.  Ideal volume is 50-100µL of combined Cas9 mRNA, sgRNA, and donor if applicable.
  • If using a donor, allow sgRNA and Cas9 mixture to sit at room temperature for 10 minutes prior to adding the donor.
  • Centrifuge vials at >10,000xg for 10-15 minutes.Take top 25µL of supernatant and transfer to a new microtube. The reagents are now ready for injection.
  • Reference your CofA or contact Horizon for suggested concentrations

 

Standard Reagent used for Transfection into cells preparation:

Recommended storage: -80℃

Preparation instructions:
  • Thaw each vial of Cas9 (mRNA or RNP) and sgRNA and donor if applicable and immediately place on ice.
  • Make appropriate dilutions (if necessary) to obtain desired final transfection concentrations and volume using provided buffer.
  • Reference the the transfection reagent's recommended protocol for introduction of CRISPR-Cas9 Reagents into cells and follow accordingly.

 

Micro-injection technical troubleshooting:

My prepped RNA is sticky/viscus/contains debris/ too many embryos are lysing when I try to inject. How can I fix this?
  • First try enlarging the opening of your needle (i.e. break the tip against the holding pipette).
  • Second, if that does not help spin the sample down in the MI ready vial at 10,000rpm (or top speed) for 10 minutes. Use only the top layer to load a new needle. Try spinning and reloading at least two times.
  • Third, proceed to a new Micro-injection ready aliquot if issues persist. Repeat steps 1 and 2 with new aliquot if issues remain.

 

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