Plasmid stability in a liquid culture

A liquid culture can be gloriously cloudy and still be the wrong cells. The decision this glossary supports is whether the flask you are about to miniprep still holds the plasmid, or only the descendants that threw it away. Growing and transforming the strain is covered in bacterial culture and transformation. How the insert was built and checked is plasmid cloning from insert to colony. The drug you rely on to keep the plasmid is explained in antibiotic selection and blue-white screening.

Medium and antibiotic classes are specified from the reagents and chemicals catalogue, on the molecular biology pathway. A question about the insert sequence can be framed with the custom gene synthesis reference. Use the quote request when you need a host or a marker specified in writing. A method can be discussed.

Cloudy means biomass

Optical density and a thick pellet report how many cells you grew. They do not report how many of those cells carry the circle of DNA you care about. Plasmid DNA is a small fraction of the biomass even when every cell has it. When most cells have lost it, the prep fails quietly: genomic background, no supercoiled band, a nanodrop number that was mostly something else. The vocabulary below is how you talk about that failure without blaming the kit first.

Public cloning language is summarised in the Addgene molecular biology reference. Protein products of marker genes can be looked up as sequences in NCBI Protein, which tells you what the resistance protein is, not whether your flask still contains it.

Segregational stability

Segregational stability is the inheritance of the plasmid at division. A cell with a hundred copies will usually hand some copies to both daughters even without a dedicated partitioning system. A cell with one or two copies will, at a measurable rate, produce a daughter with zero. That daughter is a plasmid-free segregant. It is not a mutant you did anything dramatic to create. It is the arithmetic of low copy number.

Once a plasmid-free segregant exists, its future depends on growth rate. If the plasmid is nearly free to carry, the segregant may stay rare for many generations. If the plasmid is a burden, the segregant doubles faster and takes over the flask. Liquid culture is where that takeover is invisible. On a plate with antibiotic, the segregant would not have formed a colony. In broth without antibiotic, it becomes the pellet.

Plasmid burden

Burden is the extra work the plasmid imposes: DNA to replicate, marker protein to make, and any insert that is expressed when you wish it were quiet. A strong promoter leaking a toxic product is a large burden. A big, high-copy empty vector can be a milder one and still matter over a long culture. Burden is why "it grew fine" is not evidence of stability. The cells that grew finest may be the ones that quit the plasmid.

Lower temperature, a lower-copy origin, or a tighter promoter are structural answers when a construct is known to be hard to keep. They are chosen at the design stage. They are not restored by shaking a lost culture harder.

Antibiotic selection in liquid

Selection in liquid means the drug is in the broth at a level that stops plasmid-free cells, for the whole time you grow them. It only works while three things remain true. The molecule matches the marker. The molecule is still chemically active. The marker is being expressed. A bla plasmid in kanamycin broth is not under selection. An ampicillin broth that sat warm all week may not be either. A culture so dense that a beta-lactam has been destroyed by the enzyme from the resistant cells can also lose selection late in growth: the drug is gone, and any segregant born in that last stretch is free to expand. That is one reason a fresh colony into a fresh selective broth is safer than serial dilution of an old flask into another old flask.

The Addgene protocol for inoculating a culture starts from a colony for this reason. A loop from a mixed liquid is how you preserve the takeover.

A toxic insert

A toxic insert is a sequence the host grows better without. Under antibiotic, cells often cannot afford to lose the whole plasmid, because the marker is on it. They can afford to delete the insert, invert a promoter, or pick a mutation that stops expression. You still have resistance. You no longer have the gene. The gel may show a smaller plasmid. A digest that should have cut out a fragment shows the parental backbone instead. Sequence is the confirmation. Turbidity will not hint at this. The culture looks healthy precisely because the toxin is gone.

Do not keep "the fast flask" and throw away "the slow flask" when the insert is known to be hard on the cell. Speed can be the deletion.

Terms and the result you should expect

How a prep goes empty

Start from a single colony that grew on fresh selective agar. Inoculate selective broth. Do not passage a non-selective overnight "because it was already dense". If the miniprep fails, restreak the glycerol stock or the colony plate onto antibiotic again. Growth there means the stock still has the marker. Failure there means you archived the segregant. A digest of an unexpected size means the marker survived and the insert did not. Sequence before you repeat the expression.

A control plasmid you trust, grown in parallel with the same antibiotic lot, separates a bad drug stock from a bad construct. If the control yields plasmid and yours does not, the broth recipe is innocent. If neither yields plasmid, the antibiotic in that bottle is the prime suspect.

Institutional limits stay where they were

Nothing about plasmid loss changes the organism. These are K-12 cloning strains under the containment already set. A culture that lost its plasmid is still a live culture. Inactivate it. Do not tip it out because the gel was blank. This page is not medical advice and not a method for maintaining a virulence plasmid or any construct outside the approved list. The NCBI Bookshelf is a public molecular-biology reference for the cell biology behind division and growth. It is not an approval to change host.

A flask that sat through a power cut

When the shaker stops, aeration stops and growth becomes a stress. Cells that drop a burden can be the ones that cope. When the power returns, the culture may look recoverable and be enriched for segregants. Do not miniprep it and call the band "the clone" if a band even appears. Go back to a selective plate. In a hot room, ampicillin in a broth that is already dense is particularly easy to exhaust. A sister flask with a more stable marker, where the map allows, is a diagnostic, not a casual swap of drugs.

What to specify

Name the host, the origin of replication if you know it, the marker, the insert's tendency to slow growth, and whether cultures must be started from single colonies on selective agar. Ask for medium and antibiotic specifications that match that marker. The quote request carries that note. A method can be discussed. The custom gene synthesis reference is appropriate when the burden is the sequence you are still designing. Read it as a prompt for the enquiry, not as a claim that the gene is already in production.