Table 1 Protocol troubleshooting guide: Common problems, causes and solution
From: CRISPR-mediated generation and comprehensive phenotyping of Duchenne Muscular Dystrophy mouse models
Protocol | Step | Problem | Possible Reason | Solution |
|---|---|---|---|---|
In vitro Transcription (IVT) of Guide RNAs for Cytoplasmic Microinjection | 15 | Unable to get founders with desired deletion | Bad quality of Cas9 mRNA | Repeat Cas9 mRNA IVT or source Cas9 mRNA from reputable source; use alternative protein form of Cas9 in microinjection |
Bad quality of one or both gRNAs | Re-visit guide RNA design and the oligos ordered. Repeat IVTs. Alternatively, change to different gRNAs | |||
Microinjection technical issue | Ensure that the microinjectionist possesses adequate skill and experience in the technique | |||
Forelimb Grip Strength testing | 6 | Large variation in grip strength values | Technical issues by the operators | Ensure mouse handling and testing conducted by the same and experienced operator |
Dystrophin Immunofluorescence | 4 | Unable to observe dystrophin immunofluorescence | Over-fixation | Skip fixation or perform fixation post slicing with optimised fixation reagents and duration |
Western Blotting | 7 | Degraded Protein | Inadequate protein extraction or storage | Use fresh tissue Add protease inhibitors Keep samples on ice Avoid freeze–thaw cycles |
Postmortem Tissue Processing | 7 | Poor tissue morphology | Improper freezing | Ensure the muscle section is not stretched when snap freezing Keep sample sizes consistent for uniform freezing Optimise the time each tissue is spent suspended in isopentane according to size |