PurposeHuman expression plasmid for SpCas9 sgRNA (need to clone in spacer into BsmBI sites): U6-BsmBIcassette-Sp-sgRNA
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||65777||Standard format: Plasmid sent in bacteria as agar stab||1||$75|
This material is available to academics and nonprofits only.
Backbone manufacturerJoung lab (Addgene Plasmid # 43860)
Vector typeMammalian Expression, CRISPR
Growth in Bacteria
Growth Strain(s)XL1 Blue
Copy numberHigh Copy
Gene/Insert nameSpCas9 gRNA backbone, without spacer sequence
- Promoter U6
- Cloning method Gibson Cloning
- 5′ sequencing primer OS280 (5'-CAGGGTTATTGTCTCATGAGCGG-3') (Common Sequencing Primers)
Terms and Licenses
- Not Available to Industry
- Zeocin® is an InvivoGen trademark.
Use BsmBI sites to insert sgRNA spacer sequence into the vector. See the supplemental document "Joung Lab gRNA Cloning Protocol" for more details.
This plasmid has been found to be somewhat unstable and prone to concatenation. Concatenation often does not impact plasmid function, but may reduce transformation efficiencies. You may need to screen multiple colonies to isolate the monomeric version of this plasmid. If you still have trouble isolating the monomeric version, you might consider linearizing, gel extracting, re-ligating, and transforming the plasmid.
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:BPK1520 was a gift from Keith Joung (Addgene plasmid # 65777 ; http://n2t.net/addgene:65777 ; RRID:Addgene_65777)
For your References section:Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK. Nature. 2015 Jun 22. doi: 10.1038/nature14592. 10.1038/nature14592 PubMed 26098369