PurposeFor in vitro expression and purification of Cas9 protein
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||47327||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
- Backbone size w/o insert (bp) 5368
Vector typeBacterial Expression, CRISPR
Growth in Bacteria
Growth instructionsFor expression and purification of Cas9, transform into Rosetta strain.
Copy numberHigh Copy
Insert Size (bp)4100
/ Fusion Protein
- His (C terminal on backbone)
- Cloning method Restriction Enzyme
- 5′ cloning site NcoI (not destroyed)
- 3′ cloning site NotI (not destroyed)
- 5′ sequencing primer T7 (Common Sequencing Primers)
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.
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 ; http://n2t.net/addgene:47327 ; RRID:Addgene_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