CMV-CAS9-2A-GFP Plasmid

CRISPR Human - Deletion TRIB1

Experiment
CRISPR Human - Deletion TRIB1
Product
CMV-CAS9-2A-GFP Plasmid from Sigma-Aldrich
Manufacturer
Sigma-Aldrich

Protocol tips

Upstream tips
Sigma CRISPR plasmid products are delivered as mini-prep aliquots,
which may not be suitable for transfection into particular cell types. For best results, use maxi-prepping plasmids using endotoxin-free DNA purification kits prior to transfection

Publication protocol

CRISPR-Cas9 induced disruption of chromosomal TRIB1 locus
HepG2 cells were plated in a 6 well plate (400,000 cells/well) in growth medium and after overnight incubation they were transfected using X-tremeGENE HP DNA transfection reagent (Roche) with 1.5 μg CRISPR-Cas9 plasmid (pCMV-Cas9-GFP, Sigma) and 1.5 μg of the repair template DNA fragment. The CRISPR-Cas9 plasmid carried the target sequence 5’- GGATACACGCTTCGGCCTTCTC-3’ in the guide RNA gene. The repair template consisted of three segments that were individually PCR amplified using HepG2 genomic DNA as a template for the TRIB1 target flanking segments and pCINeo plasmid as a template for NeoR cassette segment. The sequences of the PCR primers were designed to provide an overlap with the adjacent segments to allow for final PCR extension and amplification of the entire repair template.

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Reviews

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Discussion

Discussion

1 year ago

Author: Milena Alexeyeva Russian Federation

DNA insert using CRISPR

I would like to excise a large strand of DNA and insert a new one using CRISPR. My problem is that my strand will be a little over 1kb and I am not sure if this is going to be a limiting factor. Also, how long should the homology arms be for a region of this size?

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Papers

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for CMV-CAS9-2A-GFP Plasmid below.

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Videos

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