Guide for CRISPR Gene Knockout
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The CRISPR Cas9 system is easily one of the biggest biotech breakthroughs of this century. With its proven efficiency, speed, and relative ease-of-use, this system is the most accessible tool for everyday labs to conduct gene editing projects. To help you understand the versatility of the CRISPR Cas9 system, we have compiled an introductory guide that covers all you need to know to begin using CRISPR Cas9 for gene knockout experiments in a mammalian system. Simply fill in the form below to download our free knockout manual.
The following guide covers:
Table of Contents
- 04 A. CRISPR Products and Services Offered by abm
- 05 B. Introduction to CRISPR Technology
- 06 i. The Story of CRISPR’s Discovery and Development
- 08 ii. Why CRISPR is a Cut Above Other Gene Editing Tools
- 10 C. CRISPR Knockout Protocol at a Glance
- 11 D. sgRNA Design Considerations
- 12 i. sgRNA Target Validation
- 13 ii. Plasmid Amplification Protocol
- 13 1. Transformation
- 14 2. Plasmid Purification (Miniprep)
- 14 3. Plasmid Confirmation (Restriction Enzyme Digest)
- 15 E. sgRNA and Cas9 Delivery into Target Cells
- 17 i. Transfection and Transduction Procedure
- 17 1. Transfection Procedure
- 18 2. Packaging and Transduction Procedure
- 19 3. Killing Curve Determination
- 20 4. Drug Selection
- 22 F. Validation of CRISPR Gene Editing
- 24 i. gDNA PCR Primer Design
- 25 ii. Protocol for Using the CRISPR Genomic Cleavage Detection Kit (Cat. No. G932)
- 25 1. Cell Lysis
- 25 2. PCR Amplification
- 27 3. Gel Analysis of Cleavage Products
- 28 iii. Protocol for Using Sanger Sequencing to Validate CRISPR Gene Editing (Cat. No. C199)
- 28 1. CRISPR Sample Preparation for Sanger Sequencing
- 29 2. Analyzing Sanger Sequencing Results
- 30 iv. Next Generation CRISPR Validation (Cat. No. IA00100)
- 30 v. Western Blot Validation
- 31 G. CRISPR Knockout Case Study
- 34 H. Conclusion
- 35 I. References
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