Purpose(Empty Backbone) Cas9 from S.pyogenes with CMV-mcherry cassette, and cloning backbone for sgRNA
|Item||Catalog #||Description||Quantity||Price (USD)|
|Plasmid||98750||Standard format: Plasmid sent in bacteria as agar stab||1||$65|
This material is available to academics and nonprofits only.
Backbone manufacturerZhang lab (Addgene plasmid #42230)
Modifications to backboneCMV- mcherry-pA amplified from mCherry-C1 (clontech) and inserted in to pX330 in NotI site
Vector typeMammalian Expression, CRISPR
- Promoter U6
Growth in Bacteria
Growth Strain(s)NEB Stable
Copy numberHigh Copy
- Cloning method Restriction Enzyme
- 5′ sequencing primer CATCGTGGAACAGTACGAA
- 3′ sequencing primer CGTTCACGGAGCCCTCCA (Common Sequencing Primers)
Because there exists a BbsI cleavage site on CMV-mcherry-pA cassette as well, it is suggested that the BbsI digested product need to be recovered directly instead of agarose gel purification.
Digest the px330-mcherry plasmid with BbsⅠ enzyme. After that, run a small amount of the digest on an agarose gel electrophoresis to check the digestion efficiency. If the px330-mcherry plasmid has been well digested, recover the remaining digestion products using a DNA purification kit (such as Tiangen, DP214-03) for restriction enzyme cleanup. In our opinion, any commonly used DNA or PCR purification kit procedure would work well, but not agarose gel purification. We do not suggest combined digestion and ligation. For all other steps (including ligation of annealed oligos into this plasmid), refer to the Zhang lab protocol for pX330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene plasmid #42230).
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:px330-mcherry was a gift from Jinsong Li (Addgene plasmid # 98750)
For your References section:Correction of a genetic disease in mouse via use of CRISPR-Cas9. Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, Yan Z, Li D, Li J. Cell Stem Cell. 2013 Dec 5;13(6):659-62. doi: 10.1016/j.stem.2013.10.016. 10.1016/j.stem.2013.10.016 PubMed 24315440