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CAS9P Sigma-Aldrich

Cas9 plasmid

  •  NACRES NA.51

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Properties

Related Categories CRISPR-Cas9, Cas9-only (DNA, mRNA, and virus), Functional Genomics and RNAi, Molecular Biology
Quality Level   200
recombinant   expressed in E. coli
packaging   vial of 50 μL
concentration   20 ng/μL in TE buffer; DNA (1μg of plasmid DNA)
Promoter   Promoter name: CMV
selection   kanamycin
shipped in   dry ice
storage temp.   −20°C

Description

General description

The Cas9 expression plasmids use the CMV promoter for strong transient expression of Cas9. Alternate promoters can be substituted by replacement of CMV using MluI and NheI. Also, the Cas9 expression plasmids can be linearized using XbaI for T7-based mRNA production.

Application

Functional Genomics/Target Validation
• Creation of gene knockouts in multiple cell lines
• Complete knockout of genes not amenable to RNAi
• Creation of knock-in cell lines with promoters, fusion tags or reporters integrated into endogenous genes

Components

1 vial containing 1ug of Cas9 plasmid.

Please note, product does not contain guideRNA sequence. This must be purchased separately through the Custom CRISPR product tab.

Principle

CRISPR/Cas systems are employed by bacteria and archaea as a defense against invading viruses and plasmids. Recently, the type II CRISPR/Cas system from the bacterium Streptococcus pyogenes has been engineered to function in eukaryotic systems using two molecular components: a single Cas9 protein and a non-coding guide RNA (gRNA). The Cas9 endonuclease can be programmed with a single gRNA, directing a DNA double-strand break (DSB) at a desired genomic location. Similar to DSBs induced by zinc finger nucleases (ZFNs), the cell then activates endogenous DNA repair processes, either non-homologous end joining (NHEJ) or homology-directed repair (HDR), to heal the targeted DSB.

Physical form

Sigma Cas9 plasmid DNA is supplied at concentrations of 20ng/ul in 50ul.

Preparation Note

Sigma CRISPR plasmid products are delivered as mini-prep aliquots, which may not be suitable for transfection into particular cell types. For best results, we advise maxi-prepping plasmids using endotoxin-free DNA purification kits prior to transfection.

Other Notes

Must be used in conjunction with a U6-gRNA plasmid in order to mediate a double strand break in the DNA.

Typical transfection concentrations used in literature are in the ranges of >= 1.0 ug/uL and <= 5 uL of Cas9 plasmid combined with >= 1.0 ug/uL and <= 5 uL of U6-gRNA plasmids. (All dosages above assume 0.5 to 1 million cells nucleofected)

Legal Information

CRISPR Use License Agreement

Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport
Flash Point(F) 
Not applicable
Flash Point(C) 
Not applicable

Documents

Certificate of Analysis (COA)

Please Enter a Lot Number
Protocols & Articles

Articles

A CRISPR/Cas-GFP Vector for Rapid Expression Verification and Enrichment of Genome Edited Cells

In many genome editing experiments involving ZFNs and CRISPR/Cas nucleases, the first challenge is achieving successful delivery of plasmids and subsequent expression of the encoded nucleases. While ...
Keywords: Cloning, Gene expression, Sequencing, Transfection

CRISPR/Cas Nuclease RNA-guided Genome Editing

What is CRISPR/Cas9? How does CRISPR/Cas work? How does CRISPR Cas9 work? What is CRISPR/Cas9? Our role in developing CRISPR/Cas9
Keywords: Acetylations, Catalysis, Cell culture, Degradations, Gene expression, Genetic, PAGE, Polymorphisms, Transcription, Transduction, Transfection

Creating Transgenic Mice using CRISPR-Cas9 Genome Editing

While several genome editing tools have been developed in recent years, including zinc finger-based strategies and TALENs (transcription activator-like effector nucleases), none have been as efficien...
Keywords: Amplification, Cell culture, Enzyme-linked immunosorbent assay, Gene expression, Genetic, Genetics, Immunohistochemistry, Immunology, Nutrition, Polymerase chain reaction, Recombination, Southern blot, Transcription, Transfection, Western blot

Genome Editing in Plants with CRISPR/Cas9

Successful ZFN-induced gene targeting was published as early as 2003. Since that time targeted genome editing technology has rapidly advanced and been made commercially available. Most recently, the ...
Keywords: Cloning, Functional genomics, Gene expression, Genetic, Genomics, PAGE, Plant biotechnology, Recombination, Transcription, Transfection, transformation

Tips for Cell Engineering using Cas9-GFP CRISPR plasmids

CRISPR endonucleases have shown wide variation in their activity, even among multiple CRISPRs designed within close genomic proximity.1  For this reason, we highly recommend that you test 3 to 4 CRIS...
Keywords: Cell culture, Cloning, DNA purification, Gene expression, Microscopy, Purification, Reductions, Sequencing, Transcription, Transfection

Related Content

CRISPR

In recent years CRISPR has revolutionized gene editing capabilities, leading to sophisticated ways to create success with any experiment. As the first company to offer custom biomolecules globally fo...
Keywords: Genetic, Genomics, Transduction, Transfection

Predictive Models for Neuroscience using CRISPR [VIDEO]

Caroline Beckett, the global CRISPR product manager, discusses reagent solutions for creating predictive models for neuroscience research. She notes that many neuroscientists want to be able to creat...
Keywords: Cell culture, Diseases, Neurodegenerative Diseases, Neuroscience

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