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
Expected results
Gel electrophoresis results are expected to resemble the gel image below.
This image represents results obtained after a 20 minute run on a GELATO™ electrophoresis system
at 135 V.
Note: If you wish to skip the pre-lab activity to predict the band sizes, you may provide these
expected band sizes to the students.
Interpretation
• Tubes A and B will both result in DNA that remains uncut and 3,142 base pairs long. Both tubes
serve as different examples of a negative control.
° Tube A is missing both the Cas9 nuclease and the gRNA needed to form a functional Cas9/
gRNA complex.
° Tube B contains Cas9 but is missing the gRNA needed to form a functional Cas9/gRNA
complex.
° Together, these two tubes demonstrate that the Cas9 nuclease on its own is insufficient to
target and cut DNA.
• Tube C should show that the DNA sample was cut by Cas9.
° Tube C contains Cas9 nuclease and gRNA1. This forms a Cas9/gRNA complex that cuts at
the sequence targeted by gRNA1.
° gRNA1 targets the DNA sample at base pair 1,341, resulting in a band of 1,341 bp and a band
of 1,801 bp when cut by Cas9.
° You may also continue to see a band at 3,142 bp. This represents residual uncut DNA. See
below for more discussion of partial cutting.
AH BH CH DH + -
Note: You may see faint bands that are significantly larger than the top band of
the DNA ladder. These bands represent extraneous bacterial DNA and can be
ignored for the purposes of this experiment.
