miniPCR® Knockout! A CRISPR/Cas Gene Targeting Lab

Perform cutting-edge genome editing!

• Techniques: Micropipetting, bacterial transformation, gene targeting
• Topics: Genome editing, biotechnology, molecular biology
• Time required: Requires a 2-hour class period
• Level: Advanced high school through college

Become a genetic engineer and use CRISPR/Cas to disable a gene in bacteria. Scientists use the CRISPR/Cas system to alter the DNA in living cells. Now you can bring this cutting-edge technique to your advanced biology class! In this lab, E. coli cells which express the lacZ gene from a plasmid will be transformed with another plasmid containing the cas9 gene and a custom guide RNA. This guide RNA contains a sequence complementary to the lacZ gene so, in transformed cells, the guide RNA will target Cas9 to cut the lacZ gene, disabling it. Students can easily identify their CRISPR/Cas knockouts using the color of the bacterial colonies. Cells which express lacZ can break down the chemical X-gal into a blue product. Cells with a functional lacZ will be blue, while cells in which the CRISPR/Cas system has successfully disabled lacZ will revert to a white phenotype.

Required Equipment:
- Micropipettes and tips
- Water bath or dry bath
- Incubator

Storage Notes:
- Some reagents require refrigerator storage and some require freezer storage
- Reagents are stable for 3 months when stored as directed

Note: This is a technically advanced lab that requires a 2-hour class period. Students should have prior experience with bacterial transformation.

Accessories information: The Learning lab companion kit (catalog no. 470352-026) contains all the consumables (except water) needed for a class of 32 students. These reagents and consumables can also be sourced independently.

Delivery information: This lab provides enough reagents for eight lab groups: plasmid DNA; pCtrl DNA; pKO DNA; calcium chloride; lyophilized DH5alpha E. coli with lacZ plasmid; LB + ampicillin; SOC recovery medium; ChIX Mix; LB agar; 20 sterile petri dishes; 25 sterile spreaders; 100 sterile 1.7 ml tubes; Knockout! sticker.