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

Ordering

Item Catalog # Description Quantity Price (USD)
Plasmid 47327 Plasmid sent as bacteria in agar stab 1 $65

This material is available to academics and nonprofits only.

Backbone

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

Growth in Bacteria

  • Bacterial Resistance(s)
    Kanamycin
  • 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
  • (Common Sequencing Primers)

Resource Information

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.

How to cite this plasmid ( Back to top)

These plasmids were created by your colleagues. Please acknowledge the Principal Investigator, cite the article in which the plasmids were described, and include Addgene in the Materials and Methods of your future publications.

  • For your Materials & Methods section:

    pET-28b-Cas9-His was a gift from Alex Schier (Addgene plasmid # 47327)
  • For your References section:

    Efficient mutagenesis by Cas9 protein-mediated oligonucleotide insertion and large-scale assessment of single-guide RNAs. Gagnon JA, Valen E, Thyme SB, Huang P, Ahkmetova L, Pauli A, Montague TG, Zimmerman S, Richter C, Schier AF. PLoS One. 2014 May 29;9(5):e98186. doi: 10.1371/journal.pone.0098186. eCollection 2014. PONE-D-14-14210 [pii] PubMed 24873830