miniPCR bio
TM
Chopped! Using CRISPR/Cas9 to cut DNA - Instructor's and Student's Guide
Version: 1.0 - Release: May 2022 - © 2022 by miniPCR bio™
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Instructor's Guide
Troubleshooting
These in vitro CRISPR reactions use sensitive reagents and require adherence to the described
protocol. All of the following can adversely affect the DNA cutting efficiency:
Reagent storage
The Chopped! reagents will be shipped overnight on ice but will need to be stored in the freezer (at
least -20 °C) immediately upon arrival.
• Tip: 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).
Cas9 Nuclease handling
The Cas9 Nuclease reagent is especially sensitive to temperature fluctuations. It is shipped in a
concentrated format for maximal stability. The most common source of failure in this activity is the
degradation of the Cas9 Nuclease, resulting from poor handling.
• Tip 1: Keep the Cas9 Nuclease on crushed ice at all times.
• Tip 2: Please dilute the Cas9 Nuclease as close to the start of the activity as possible. If you
must dilute and dispense the Cas9 Nuclease more than 12 hours in advance, dilute the Cas9
Nuclease and store aliquoted tubes in the freezer for no more than a week. Thaw the diluted
aliquots of Cas9 Nuclease on ice just before use. The rest of the reagents may be thawed,
handled, and distributed at room temperature.
• Tip 3: All of the Cas9 Nuclease in the supplied tube should be diluted at once. Add Nuclease-
Free Water directly to the 5X Cas9 Nuclease tube as directed on page 8. Diluting just a
portion of the Cas9 Nuclease and pipetting the concentrated Cas9 Concentrate can lead to
inconsistent results.
Mixing the reactions
Thorough mixing of the reagents in the reactions is essential for the successful cutting of the DNA.
• Tip: Please have students mix their reactions as indicated in the protocol before EACH of the
incubation steps (i.e., mix after combining Cas9 Nuclease and gRNA, mix after adding DNA
sample, and mix after adding the Proteinase K Solution). The suggested method of mixing
is with a tube vortexer for five seconds, but if you do not have a vortexer, you may instead
pipette up and down 10 times to mix.
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