Cas9 RT-PCR primer set

Quality level: 200

Recombinant: expressed in E. coli

Packaging: 50 μL vial

Concentration

20 ng / µL in TE buffer; DNA (1 μg of plasmid DNA)

Promoter

Promoter name: CMV

Selection: kanamycin

Sent in: dry ice

Storage temperature: −20 ° C

General description

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

Application

Functional Genomics / Target Validation

  • Creation of gene knockouts in multiple cell lines.
  • Complete knockout of RNAi non-susceptible genes
  • 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 that the product does not contain a guide RNA sequence. This must be purchased separately through the custom CRISPR product tab.

Beginning

CRISPR / Cas systems are used by bacteria and archaea as a defence against invading viruses and plasmids. Recently, the type II CRISPR / Cas system of the bacterium Streptococcus pyogenes has been designed 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, targeting a DNA double-stranded break (DSB) at a desired genomic location. Like zinc finger nuclease (ZFN) -induced DSBs, the cell activates endogenous DNA repair processes, either non-homologous end-junction (NHEJ) or homology-directed repair (HDR), to cure the DSB objective.

Physical form

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

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 recommend maximal plasmid preparation using endotoxin-free DNA purification kits prior to transfection.

Other notes

  • It must be used in conjunction with a U6-gRNA plasmid to mediate a double-stranded break in DNA.
  • Typical transfection concentrations used in the 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 the above doses assume 0.5 to 1 million nucleofected cells)

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