Strains and Culture conditions
V. cholerae strains used in this study are derived from E7946. Bacteria were routinely grown on LB agar plates and in LB broth with aeration at 37°C. Antibiotics were supplemented as appropriate at the following concentrations: 75 μg/ml kanamycin, 100 μg/ml spectinomycin, 1.25 or 2.5 μg/ml ch...
Zebrafish husbandry
All vertebrate animal work was performed at the facilities of the University of Utah, CBRZ. This study was approved by the Office of Institutional Animal Care & Use Committee (IACUC # 18-2008) of the University of Utah’s animal care and use program.
Cloning and transcription of CRISPR-Cas systems and a...
Sam
12 months ago
Materials & Methods
Bacteria and growth conditions
Bacteria (Table S4) were cultured in lysogeny broth36 at 37°C. pCA24N-based plasmids37 were retained in overnight cultures via chloramphenicol (30 μg/mL), and kanamycin (50 μg/mL) was used for deletion mutants, where applicable.
Spacer knockout
To attempt to delete the CRISPR-Cas spacer reg...
Amos Fung
over 1 year ago
Cas9 Condition Optimization
This protocol runs several in vitro Cas9 cleavage conditions for identifying the optimal setup for a specific target (in this case the Sub1 gene).
Cas9 Cleavage
Cas9 nuclease reactions can be set up as described in the table below. For identifying cleavage conditions, use a combination of 1X and 2X Cas9 reactions along wi...
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