All products have special prices for bulk purchase, please contact for more details if required.

Cat. No.: SGRCK-10 (for 10T)

Description

The Surrogate GFP Reporter Construction Kit, also known as the Gene-editing Surrogate GFP Reporter Construction Kit, is developed based on surrogate reporter gene technology. It is designed to construct GFP surrogate reporter plasmids that can effectively trace or enrich cells undergoing successful gene editing.

Typically, the efficiency of gene editing in cells is not very high, and only a portion of the cells undergo gene editing. However, there is a lack of effective methods for live-cell tracing of which cells have been edited. This kit uses surrogate reporter gene technology, allowing users to design and synthesize gRNA-targeted genomic DNA sequences (annealed from synthesized single-stranded DNA) according to the instructions. These sequences are then ligated to the linearized vector provided in the kit to construct surrogate reporter plasmids for gene-editing cell tracing or enrichment. When the surrogate reporter plasmid is co-transfected with Cas9 and sgRNA expression plasmids into mammalian cells, the presence of GFP fluorescence signals in the cells can be used to determine if effective gene editing has occurred. Subsequently, GFP-positive cells can be sorted using flow cytometry, obtaining a high proportion of gene-edited cells for further experiments. Since the surrogate reporter gene plasmid contains DNA sequences identical to the target genome, when the surrogate reporter gene undergoes a frameshift mutation leading to GFP expression, the genomic DNA in these cells is also likely to have been edited. This allows for the determination of gene editing in cells based on GFP fluorescence signals and the preliminary assessment of the likelihood of genomic DNA editing.

This kit, based on surrogate reporter gene technology, involves the insertion of an exogenous fragment identical to the target gene sequence into the pre-designed vector provided in the kit. The vector contains a GFP reporter gene that is only expressed when gene editing induces a frameshift mutation. When the constructed surrogate reporter plasmid is co-transfected with plasmids expressing Cas9 and sgRNA, green fluorescence directly indicates the occurrence of gene editing in the cells. Therefore, these reporter genes are named surrogate reporters. Research has shown that when a target sequence on one chromosome is mutated by nucleases like CRISPR/Cas9, TALEN, or ZFN, the same target sequence on the homologous chromosome in the same cell is more likely to mutate. Based on this principle, flow cytometry can be used to enrich GFP-positive cells, significantly increasing the probability of obtaining gene-edited cells.

The Linearized pCMV-RFP-STOP-GFP provided in this kit comprises genes encoding two fluorescent proteins (RFP and GFP). The sequence between RFP and GFP includes the target sequence for sgRNA, the PAM sequence, and a stop codon.

Components

• Linearized pCMV-RFP-STOP-GFP
• Ultrapure Water
• Quick Ligase
• 10X Quick Ligation Buffer
• Primer-F

Features

• Highly Efficient: This kit includes an ultra-fast DNA ligase, enabling ligation in just 5 minutes at 25ºC. This results in a high number of colonies in subsequent experiments, with a cloning positive rate of up to 100%. 
• Convenient Use: The kit provides a linearized vector, allowing users to anneal and ligate the designed target-specific DNA oligos according to the instructions, and then proceed with transformation and plating. No additional vector processing steps are required, making the process fast and convenient.
• Specific Use: This kit is intended for the rapid construction of surrogate reporter plasmids only and does not include any plasmids encoding Cas9 or sgRNA. For rapid construction of plasmids encoding Cas9/sgRNA, it is recommended to use the Quick Construction Kit (Puro).

Storage

Store at -20ºC for up to two years.

Precautions

• The surrogate reporter plasmids constructed with this kit contain RFP and GFP, with GFP serving as the reporter gene. Therefore, plasmids encoding Cas9 or sgRNA co-transfected with these should not contain GFP or other fluorescent proteins with similar excitation/emission spectra, such as YFP.
• For routine transformation of E. coli, there is no need to purify the ligation product before transformation. However, when using electroporation, it is advisable to purify the DNA using a purification kit or phenol-chloroform extraction before electroporation.
• Gel electrophoresis is not necessary for routine ligation reactions. If you need to observe the ligation products via gel electrophoresis, incubate at 65ºC for 10 minutes to inactivate Quick Ligase, avoiding band shift caused by Quick Ligase binding to DNA.
• This product is intended for scientific research by professionals only. It is not for clinical diagnosis or treatment, nor for use in food or medicine. Do not store it in common household environments.
• For your safety and health, wear a lab coat and disposable gloves when handling.

Only for research and not intended for treatment of humans or animals

Journals Using SBS Genetech Products                                       Universities Using SBS Genetech Products

SBS Genetech is a long-term sponsor of Cold Spring Harbor Laboratory