Description
Use PCR to confirm your CRISPR/Cas gene Knockout!
(This is an optional follow-up experiment to the Knockout! CRISPR/Cas Lab)
After completing the Knockout! Lab, bacteria transformed with the pKO plasmid (encoding Cas9 and a guide RNA that targets the lacZ gene) should appear phenotypically white, whereas bacteria transformed with pCtrl plasmid (encoding Cas9 and a random guide RNA) should appear phenotypically blue. Use PCR to determine the genotypes of the bacteria and confirm that you successfully used the CRISPR/Cas system to disrupt the lacZ gene.
- Techniques: Micropipetting, PCR, gel electrophoresis
- Topics: Genome editing, molecular biology, biotechnology
- Time required: Requires two 45-minute class periods, or a single two-hour class period
- Level: Advanced high school, college
What is included
This kit is a follow-up experiment to the Knockout! Lab (KT-1800-01 or KT-1800-03).
This lab kit provides enough reagents for eight lab groups:
- 5X EZ PCR Master Mix, Load-Ready™
- Knockout! Primer Mix
- lacZ Control DNA
- cas9 Control DNA
- Fast DNA Ladder 1
- Nuclease Free Water
Supplied by user:
Standard reagents and consumables must be supplied by the user. The Learning Lab Companion Kit (catalog no. KT-1510-01) contains all the consumables (except water or toothpicks) needed for 8 lab groups. These reagents and consumables can also be sourced independently.
Reagents and consumables supplied by user:
- Agarose (electrophoresis grade)
- DNA stain (e.g., GelGreen®)
- Gel electrophoresis buffer (e.g., 1X TBE)
- Distilled or deionized H2O (to dilute buffer concentrate – not included in Lab Companion Kit)
- Microtubes
- PCR tubes
- Toothpicks
Required equipment:
- Micropipettes and tips
- Thermal cycler
- Gel electrophoresis and visualization system
Storage notes:
- Reagents may ship at room temperature, but require freezer storage upon arrival
- Reagents are stable for 12 months when stored in freezer
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