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./23
C. Inactivate Cas9 enzyme with
Proteinase K
Proteinase K breaks down proteins and is used here to remove the
Cas9/gRNA complex from the DNA prior to running your sample on an
agarose gel.
Note: The Proteinase K Solution is green because it also contains gel
loading dye for running the sample on the gel; this will save the step of
adding loading dye later.
10. Add 4 μl Proteinase K Solution to each of the tubes
• Use a micropipette to add the Proteinase K Solution.
• Remember to change tips at each step!
• The total volume in each tube should be 24 μl after completing
this step. Your reactions should now have a dark green
appearance instead of clear.
Pro
K
11. Ask your instructor whether you will be stopping here for the day or continuing with gel
electrophoresis in the same class period
Tube A Tube B Tube C Tube D
Proteinase K Solution
4 μl 4 μl 4 μl 4 μl
TOTAL VOLUME 24 μl 24 μl 24 μl 24 μl
Option A: Stop here for the day Option B: Run your samples on the gel in the same
class period
• Store your samples in the freezer.
• In the next class period, samples can be thawed
and used immediately for gel electrophoresis.
• Note: If freezing your samples overnight, there is
no need to incubate tubes at 37 °C after adding
proteinase K as indicated on the right column,
Option B.
• Incubate your samples at 37 °C for at least 10
minutes before running your gel.
• Longer incubation times (up to 20 minutes) may
result in clearer gel results.
10
min
8+
hr
