New England Biolabs
EnGen® Mutation Detection Kit
E3321S
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We were interested in studying the role of DNA methylation in the differentiation of of CD4 T-cells. CRISPR-Cas9 approaches were used to knock-out epigenetic enzymes in primary cells and cell lines. This kit was used to determined the editing efficiency in the bulk cell population.
Gene editing
Cell lines / primary cells
The PCR conditions should be optimized to the size of your amplicon.
Cells were transfected with a TET1 targeting DNA cassette. After 48 hours of incubation, genomic DNA was extracted and the mutationregion was amplified. Following heteroduplex formation by denaturation& reannealing, DNA was digested with T7 endonuclease. Using a fragment analyzer, we were able to calculate the editing efficiency.
N/A
Works well and is user friendly.
Very expensive and a good fragment analyzer is required.
This is a good kit to determine editing efficiency in-house within a few hours and without employing sequencing techniques.