Integrated DNA Technologies
Alt-R Genome Editing Detection Kit
1075931
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I routinely use CRISPR/Cas9-based methodology to generate gene knockouts in cell lines. As a first step, I do pooled gene knockout using the lentiCRISPR v2 plasmid and test the editing efficiency using the T7 endonuclease I (T7EI) mismatch cleavage assay.
Genome editing
NIH-3T3 knockout cells (pooled)
Follow manufacturer's instructions.
Gtl3 gene was knocked out in NIH-3T3 cell line using lentiCRISPR v2 plasmid. ~500 bp amplicon was generated that included the predicted cut site near the middle of the amplicon. ~300 ng of the amplicon was subjected to duplex formation and nuclease treatment. The products were resolved on a 2% TBE-Agarose gel.
N/A
Straight forward to use and does not require PCR amplicon purification.
None.
Good assay to determine the editing efficiency of pooled CRISPR gene knockout.