Easy to Use with Troubleshooting

October 11, 2016

BlueskyReddit
University of Kansas Medical Center
Pathology
Postdoctoral Fellow

Overall

Quality of Results

Ease-of-Optimization

What do these ratings mean?
Write a Review

Company:

Thermo Fisher

Product Name:

GeneArt (TM) Genomic Cleavage Detection Kit

Catalog Number:

A24372

Designing a CRISPR cell line to evaluate effects of loss of a mitotic protein on my cell line models.

Experimental Design and Results Summary

Application

CRISPR Silencing

Starting Material

OVCAR-5 and SK-OV-3 which are human ovarian cancer cell lines

Protocol Overview

Transfected cells with gRNA and Cas9 for 48 hours. Lysed cells, PCR amplified region of interest, then performed denaturing and re-annealing steps in order to create mismatches that would be recognized and cleaved.

Tips

Confirm and optimize your primers BEFORE starting

Results Summary

I observed cleavage at my region of interest. I used pre-designed primers (Thermo Fisher CRISPR tool), and those did not map to my gene (used PrimerBLAST) which required me to do quite a bit of troubleshooting.

Additional Notes

None

Image Gallery

Summary

The Good

Kit worked, and is a pretty easy way to confirm Cas9 cleavage of your gene of interest

The Bad

Kits are expensive, not enough information included concerning pre-designed primers

The Bottom Line

Overall, I would recommend this kit for confirming cleavage of a given gene regardless of the source of your additional resources.

Join the discussion