pET-28b-Cas9-His
(Plasmid #47327)

• Purpose: For in vitro expression and purification of Cas9 protein
• Depositing Lab: Alex Schier
• Publication: Gagnon et al PLoS One. 2014 May 29;9(5):e98186. doi: 10.1371/journal.pone.0098186. eCollection 2014.

Backbone

• Vector backbone: pET-28b
• Backbone size w/o insert (bp): 5368
• Vector type: Bacterial Expression, CRISPR

Growth in Bacteria

• Bacterial Resistance(s): Kanamycin, 50 μg/mL
• Growth Temperature: 37°C
• Growth Strain(s): DH5alpha
• Growth instructions: For expression and purification of Cas9, transform into Rosetta strain.
• Copy number: High Copy

Gene/Insert

• Gene/Insert name: Cas9
• Insert Size (bp): 4100
• Tag / Fusion Protein:
  • His (C terminal on backbone)

Cloning Information

• Cloning method: Restriction Enzyme
• 5′ cloning site: NcoI (not destroyed)
• 3′ cloning site: NotI (not destroyed)
• 5′ sequencing primer: T7

Resource Information

• Articles Citing this Plasmid:
  • 27 References

Terms and Licenses

• Academic/Nonprofit Terms:
  • UBMTA
• Industry Terms:
  • Not Available to Industry

Depositor Comments

Please see the Schier lab website http://labs.mcb.harvard.edu/schier/ for detailed protocols about target site selection, sgRNA production, stop codon cassette design, Cas9 protein purification, injection and downstream analysis.

This plasmid was used to express a His-tagged Cas9 protein in E. coli and purify it by nickel-based affinity purification.The Cas9 protein and sgRNA were incubated at room temperature to form the Cas9 protein/sgRNA complex in vitro, which was then microinjected into zebrafish zygotes. See publication for more details.