Chopped! Using CRISPR/Cas9 to cut DNA
Use the CRISPR/Cas9 system in vitro to cut DNA in a tube and analyze the results with gel electrophoresis. Investigate how the Cas9 nuclease can be programmed to target virtually any DNA sequence—just by changing the guide RNA.
Watch the Video here
The introduction of CRISPR/Cas into
the biotechnology toolkit has revolutionized gene targeting and gene
editing. In this lab, students will use the CRISPR/Cas system in vitro
(in a test tube) to analyze the function of its molecular components.
They will use two different guide RNAs to direct Cas9 nuclease to cut a
DNA sample at two different locations and then analyze their resulting
DNA fragments using gel electrophoresis. This gene targeting lab will
allow students to investigate how the Cas9 nuclease can be programmed to
target virtually any DNA sequence—just by changing the guide RNA. This
activity will demonstrate how the CRISPR/Cas system is both
programmable and specific and why it is such a powerful genome editing
tool.
- Techniques: Micropipetting, gene targeting, gel electrophoresis
- Topics: Genome editing, biotechnology, CRISPR/Cas9
- Time required: Two 45-min class periods or one 90-min block
- Level: General high school through college
What is included ?
This lab provides enough reagents for eight lab groups:
- Nuclease-Free Water
- Reaction Buffer
- Cas9 Nuclease
- Guide RNAs (gRNA1 and gRNA2)
- DNA Sample
- Proteinase K Solution
- Fast DNA Ladder 3
IMPORTANT storage notes:
- The CHOPPED! reagents will be shipped overnight on ice and will need to be stored in the freezer immediately
upon arrival. We strongly recommend storing the reagents in the coldest
part of the freezer where temperature fluctuations are minimal (e.g.,
in the back of the freezer instead of the freezer door).
- The Cas9
Nuclease has greater stability when stored at higher concentrations. For
this reason, you will need to dilute the enzyme shortly before use. See
the Instructor’s Guide for more details.
- Reagents are stable for 12 months when stored according to these recommendations.
Required equipment:
- Micropipettes and tips
- 37 °C heat source (e.g., incubator, water bath, or heat block)
- Gel electrophoresis and fluorescence visualization system
- Optional: Vortex and microcentrifuge
Reagents and consumables supplied by user:
The Learning Lab Companion Kit
(catalog no. KT-1510-01) contains all the consumables (except water)
needed for 8 lab groups. These reagents and consumables can also be
sourced independently.
- Agarose (electrophoresis grade)
- DNA stain (e.g., SeeGreen™ or 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
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