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™
P./8
Instructor's Guide
Dilute 5X Cas9 Nuclease Concentrate
• Add 160 μl of Nuclease-Free Water directly to the 5X Cas9 Nuclease
Concentrate tube to create a 1X Cas9 Nuclease working solution. This
will yield 200 μl of 1X Cas9 Nuclease working solution ready to be
dispensed.
- Keep the Cas9 Nuclease on crushed ice at all times.
- Dilute all of the Cas9 Nuclease at once by adding Nuclease-
Free Water directly to the tube. Diluting a portion of the Cas9
Nuclease can lead to inconsistent results.
- Dilute the Cas9 Nuclease as close as possible to the start of the
lab activity and keep on ice at all times.
- If you must dilute and aliquot the Cas9 Nuclease more than 12
hours in advance, store the tubes containing the 1X Cas9 working
solution in the freezer for up to one week. Thaw the diluted
aliquots of Cas9 Nuclease on ice just prior to use.
Defrost tubes
Water gRNA2
DNA
Sample
Proteinase
K
Solution
Reaction
Buffer
Fast
DNA
Ladder
Water
5X
Cas9
Nuclease
160 μl
Keep the Cas9 Nuclease on ice at all times.
Thaw reagents
• Thaw tubes containing the reagents by placing them on a rack or benchtop at room
temperature.
• Note that the 5X Cas9 Nuclease Concentrate will remain liquid at -20 °C, does not need to be
thawed and should be kept in the freezer until it is needed.
