miniPCR® Knockout! PCR Genotyping Experiment

Use PCR to confirm your CRISPR/Cas gene Knockout!

• 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

(This is an optional follow-up experiment to the Knockout! CRISPR/Cas Lab: 470352-010)

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.

This kit is a follow-up experiment to the Knockout! Lab (KT-1800-01 or KT-1800-03).

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

 : 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.

 : 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.