miniPCR bio
TM
Chopped! Using CRISPR/Cas9 to cut DNA - Student's Guide
Version: 1.0 - Release: May 2022 - © 2022 by miniPCR bio™
Student's Guide
P./21
Laboratory guide
A. Create Cas9/gRNA
complex
Follow the steps below to mix the Cas9
Nuclease and the gRNA to allow the Cas9/
gRNA complex to form.
1. Label four plastic tubes
• Use a fine-tipped permanent marker to
label the tubes A-D.
2. Follow the table below to add reagents to
each of your tubes
• Use a micropipette to add each of the
reagents.
• After adding a reagent to a tube, check
it off in the table below so you know
you have already added it!
• Remember to change tips at each step!
• The total volume in each tube should be
15 μl after adding all reagents.
Use a micropipette to add each of the reagents. Remember to change tips at each step!
Tube A Tube B Tube C Tube D
Condition Control 1:
No Cas9
No gRNA
Control 2:
Cas9 only
gRNA1:
Cas9 +
gRNA1
gRNA2:
Cas9 +
gRNA2
Nuclease-Free Water
10 μl 5 μl - -
Reaction Buffer
5 μl 5 μl 5 μl 5 μl
Cas9 Nuclease
- 5 μl 5 μl 5 μl
gRNA1
- - 5 μl -
gRNA2
- - - 5 μl
TOTAL VOLUME 15 μl 15 μl 15 μl 15 μl
Buffer
Cas9
gRNA
15
min
10
min
A B C D
A B C D
A B C D
Mix Cas9, gRNA,
Reaction Buffer,
and Water
Incubate 10 min
at room temp
Incubate 15 min
at 37 °C
Mix in DNA Sample
Mix in Proteinase K
Solution
Gloves and protective eyewear should be worn for the entirety of this experiment.
Keep the Cas9 Nuclease on ice at all times.
Overview of experiment workflow
