This lab is easy for teachers to prepare, makes gene targeting accessible to students, and packs a powerful punch!
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 are able to 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.
Note: This is a technically advanced lab that requires a 2-hour class period. Students should have prior experience with bacterial transformation.
- Techniques: Micropipetting, bacterial transformation, gene targeting
- Topics: Genome editing, biotechnology, molecular biology
- Time required: Two 45-min or one 90-min class period
- Level: Advanced high school through college
For educational use only.
Kit Includes:
This lab provided enough reagents for eight lab groups:
- Plasmid DNA
- pCtrl DNA
- pKO DNA
- ChIX Mix
- Lyophilized DH5alpha E. coli with lacZ plasmid
- Calcium chloride
- LB + ampicillin
- SOC recovery medium
- 20 Petri dishes (90 mm)
- 25 sterile spreaders
- 100 sterile 1.5 ml tubes
- LB agar growth media
Storage:
- You will receive three bags. One is stored at room temperature, one requires refrigerator storage and one requires freezer storage
- Reagents are stable for 6 months when stored as directed
Required equipment and reagents, not included in kit:
- 20-200uL micropipettes and tips
- 100-1000uL micropipette and tips
- 37 °C incubator
