Cas9 RT-PCR Primer Set
Easily confirm expression of Cas9 mRNA—the wild-type nuclease, nickase, and inactivated Cas9 double mutant—with our ready-to-go Cas9 RT-PCR Primer Set.
Confirm Cas9 mRNA expression
With our ready-to-go Cas9 RT-PCR Primer Set, you can quickly and easily confirm Cas9 expression in cells or in vitro transcription assays.
The amplicon for Primer Set 1 is 219bp and amplicon for Primer Set 2 is 122bp. These primer sets allow for detection of Cas9 nuclease, Cas9 Nickase and Cas9 double mutant at messenger level. The primer sets are compatible with Cas9 mRNA expression from any SBI Cas9 SmartNuclease construct and vectors from Dr. Feng Zhang’s lab as well.
How It Works
Through careful selection of the target sequence and design of a donor plasmid for homologous
recombination, you can achieve efficient and highly targeted genomic modification with CRISPR/Cas9.
Cas9 protein—uses guide RNA (gRNA) to direct site-specific, double-strand DNA cleavage adjacent to a protospacer adapter motif (PAM) in the target DNA.
gRNA—RNA sequence that guides Cas9 to cleave a homologous region in the target genome. Efficient cleavage only where the gRNA homology is adjacent to a PAM.
PAM—protospacer adapter motif, NGG, is a target DNA sequence that spCas9 will cut upstream from if directed to by the gRNA.
The workflow at-a-glance
DESIGN: Select gRNA and HR donor plasmids. Choice of gRNA site and design of donor
plasmid determines whether the homologous recombination event results in a knock-out,
knock-in, edit, or tagging.
CONSTRUCT: Clone gRNA into all-in-one Cas9 vector. Clone 5’ and 3’ homology arms into HR
donor plasmid. If creating a knock-in, clone desired gene into HR donor.
CO-TRANSFECT or CO-INJECT: Introduce Cas9, gRNA, and HR Donors into the target cells
using co-transfection for plasmids, co-transduction for lentivirus, or co-injection for mRNAs.
SELECT/SCREEN: Select or screen for mutants and verify.
VALIDATE: Genotype or sequence putative mutants to verify single or biallelic conversion.