System Biosciences
Cas9 RT-PCR Primer Set (50ul at 5uM for each primer set)
- SKU:
- CAS9-PR-1
- Availability:
- Usually Shipped in 5 Working Days
- Size:
- 50 reactions
- Shipping Temperature:
- RT, Blue Ice, or Dry Ice
Description
Cas9 RT-PCR Primer Set (50ul at 5uM for each primer set). Cat# CAS9-PR. Supplier: SBI System Biosciences
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.
Products
Overview
How It Works
CRISPR/Cas9 Basics
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.
The system
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.
Supporting Data
See our Cas9 RT-PCR Primer Set evaluate expression of Cas9 knocked-into the AAVS1 site
Figure 1. Use of the Cas9 RT-PCR Primer set to evaluate Cas9 expression after Cas9 knock-in at the AAVS1 site. We knocked the CAS9 gene into the AAVS1 site using the DIY Cas9-expressing Cells HR Donor and the All-in-one Cas9 SmartNuclease & AAVS1 gRNA Plasmid. We then tested the resulting positive clones for relative Cas9 mRNA expression using qPCR (left panel) and analyzed the two highest-expressing clones (#8 and 17) for Cas9-mediated cleavage activity using a Surveyor Assay (right panel, arrow indicated band showing Cas9-mediated cleavage). In the Surveyor Assay, only the two lanes with the Cas9 clones showed Cas9 activity (arrow), with the non-transfected cells showing no Cas9 activity.
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