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CRISPR08 Sigma-Aldrich

CRISPR UNIVERSAL NEGATIVE CONTROL 3

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Properties

Related Categories CRISPR Controls (DNA and Virus), CRISPR-Cas9, Functional Genomics and RNAi, Molecular Biology
application(s)   CRISPR: suitable
shipped in   dry ice
storage temp.   −20°C

Description

General description

Universal negative control CRISPRs have been designed not to recognize any sequence in the human, mouse or rat genome. A single vector format is provided which includes the Cas9 expression cassette and gRNA sequence. This vector includes GFP which is co-expressed from the same mRNA as the Cas9 protein via a 2A peptide linkage and enables for tracking of transfection efficiency or enrichment in cell populations via FACS.

Application

Functional Genomics/Target Validation
• Creation of gene knockouts in cell lines
• Creation of knock-in cell lines with promoters, fusion tags or reporters integrated into endogenous genes

Features and Benefits

Allows for expression of Cas9 and GFP without specific targeting of the CRISPR/Cas9 system.

Components

1 vial containing 1ug of U6-gRNA/CMV-Cas9-GFP plasmid expressing a non-targeting guide sequence.

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 U6-gRNA/CMV-Cas9-GFP plasmid expressing a non-targeting guide sequence supplied at a concentration 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

Typical transfection concentrations used in literature are in the ranges of 2.0 to 8.0ug of the single vector expressing the guideRNA and Cas9 . (All dosages above assume 0.5 to 1 million cells nucleofected)

Legal Information

CRISPR Use License Agreement

Lentiviral and WPRE License Agreements

Safety & Documentation

Safety Information

RIDADR 
NONH for all modes of transport

Documents

Certificate of Analysis

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Making Headway
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

Sign up to receive the latest updates on CRISPR technology such as new journal articles, protocols, and product launches.
Keywords: Acetylations, Catalysis, Cell culture, Degradations, Gene expression, Genetic, PAGE, Polymorphisms, Transcription, Transduction, Transfection

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

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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, Transduction, Transfection

Predictive Models for Neuroscience using CRISPR [VIDEO]

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

Peer-Reviewed Papers
15

References

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