Question (2327)
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Verify plasmid quality: Confirm the plasmid is intact, at sufficient concentration, and free of contaminants (e.g., salts, ethanol, nucleases).
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Check the host strain: Use a suitable cloning strain (e.g., recA⁻/endA⁻ such as DH5α) and confirm cells are competent and not expired.
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Confirm selection conditions: Make sure the correct antibiotic is used at the proper concentration and that plates/media are fresh.
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Review transformation conditions: Ensure the transformation method (chemical or electrocompetent) and recovery time are appropriate.
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Assess plasmid compatibility: Large, low-copy, toxic, or unstable plasmids may require specialized strains or growth conditions.
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Inspect growth conditions: Verify incubation temperature, shaking speed, and culture time.
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Check plasmid sequence/features: Look for toxic genes, strong promoters, or repeats that may reduce stability.