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
Proteinase K addition
The Cas9-gRNA complex can remain bound to the DNA sample after cutting. This may cause the
results to run on the gel as a smear, rather than as distinct bands.
• Tip: Be sure to add the Proteinase K Solution to the reactions as indicated by the protocol. The
Proteinase K needs time to remove the Cas9-gRNA complex, so either incubate the reactions
at 37 °C for at least 10 minutes prior to gel analysis or store the reactions in the freezer
overnight until thawing for gel analysis. Reactions stored in the freezer do not need to incubate
at 37 °C.
High molecular weight DNA bands
You may notice bands on your gel that are significantly higher than the top band of the DNA ladder.
These bands represent extraneous bacterial DNA. They can be ignored for the purposes of this
experiment.
• Tip: Running the gel out longer or loading less of your reaction onto the gel will result in fainter
less noticeable high molecular weight DNA.
