Antibiotic selection is not proof of an insert

Antibiotic selection answers one question: could this cell divide in the presence of this drug? An insert answers a different question: which extra DNA, in which orientation, with which sequence, is on the plasmid? A colony can satisfy the first and miss the second completely. This glossary defines the four words that get collapsed into "it grew, so the clone is right," and then says what to do with the plate instead. The plating practice and the blue-white colour screen are in antibiotic selection and blue-white screening. The chain from insert to a colony worth archiving is in plasmid cloning from insert to colony.

The context is a non-pathogenic E. coli cloning strain and a plasmid that carries a resistance gene. Marker genes and antibiotics as reagent classes sit in the molecular biology catalogue.

Selection

Selection is a condition that stops the cells you do not want and lets the cells you do want form colonies. On this page the condition is an antibiotic at a dose a sensitive host cannot survive. The cell that forms a colony is the one that expressed a resistance function in time, or that escaped the drug because the drug was locally gone. Selection is powerful because it throws away the millions of cells that took up nothing. It is silent about everything else on the plasmid. People who say the selection worked are saying the drug and the marker matched. They are not saying the insert is present.

A no-DNA plate is part of the definition. If cells without plasmid grow under the same condition, you did not select. You incubated rich medium. Every later word on this page assumes the no-DNA plate was empty.

Marker

A marker is the gene whose product makes selection possible. For routine cloning that gene sits on the plasmid: a beta-lactamase gene for ampicillin and related penicillins, a phosphotransferase-class gene for kanamycin, an acetyltransferase-class gene for chloramphenicol. The map names the gene. The plate must name the matching drug. A chromosomal copy of the same marker in the host is also a marker, and it will grow whether or not your plasmid arrived. Read the genotype. A second plasmid already in the cell, kept by the same drug, is a marker too. Your new ligation is then optional.

The marker usually has its own promoter, away from the multiple cloning site. That physical separation is why insert orientation and even insert presence are invisible to selection. You can delete the cloning site entirely and the marker will still protect the cell. NCBI GenBank is one place the marker gene on a public backbone is annotated. Your tube may differ. Trust the file that matches the DNA.

Satellite

A satellite is a small colony growing in the zone around a resistant colony where the drug has been destroyed. On ampicillin plates the mechanism is specific. Beta-lactamase leaves the resistant cell, hydrolyses ampicillin nearby, and cells that are still sensitive divide in that halo. They look like extra transformants if you want them to. They are not. Many contain no plasmid. Picking one gives a culture that dies when it is diluted into fresh ampicillin, or that fails every plasmid prep.

Kanamycin and chloramphenicol do not earn the same word. Their resistance enzymes act inside the cell and do not clear a halo of agar the way leaked beta-lactamase does. A tiny colony on a kanamycin plate is not an ampicillin satellite. Call it a small colony and test it, or ignore it, but do not import the halo story. Chloramphenicol acetyltransferase is likewise the wrong mechanism for that halo. If someone tells you every antibiotic makes satellites, they are describing ampicillin and generalising the plate they know.

Satellites increase as ampicillin plates age and as colonies sit long enough to flood the agar with enzyme. Use plates that still select, and do not store a picked satellite as a glycerol stock under a construct name.

Clone

A clone, in the sense that matters after a ligation, is a colony line you are willing to treat as one plasmid sequence. Isolation is the first half: the colony came from a single cell, not from a smear of three colonies and a satellite. Identity is the second half: a check that sees the insert. Antibiotic growth supplies neither identity nor, in the satellite case, even the plasmid. Calling every resistant colony a clone is how the wrong tube gets a confident label.

The checks that promote a colony to that status are outside this glossary and inside the cloning path: a digest or a PCR that matches the map, then a sequence when the insert was copied by PCR or will be trusted. Until then the honest word is candidate. Blue-white colour, when the vector uses it, is another candidate filter. The linked screening page says why a white colony is still not an insert.

What to do with a plate once the words are straight

Pick isolated large colonies, away from halos, into fresh medium that contains the drug. Screen those cultures for the insert by a method that sees the insert. Expect some fraction to be empty vector. That fraction is not a failed definition of selection. It is selection doing only its job. If eight out of eight are empty, the join failed and more picking will not create an insert. If the plate is a lawn, remake the antibiotic. If the plate is empty, the marker, the cells or the DNA join failed, and the glossary will not tell you which until a known plasmid is transformed beside it.

Addgene's bacterial transformation notes and the molecular biology reference are public places these distinctions are used in plain language. Working concentrations still come from the plasmid map and the antibiotic note you trust, not from a single number borrowed for every drug.

Safety

An antibiotic plate is not a biosafety boundary. The strain and the insert determine containment, which your institution sets. The WHO biosafety health topic is background, not an approval. Do not treat a research colony as a diagnostic identification of a pathogen. Dispose of plates as your laboratory already requires for transformed E. coli.

Plates that lost the drug in a warm fridge

Ampicillin is not immortal in agar. Plates kept in a refrigerator that is barely cold, or left out through a long power cut in a warm building, lose activity and then grow a lawn. The lawn is not a crop of satellites and it is not a superb transformation. Date the pour, store plates at the temperature your antibiotic note requires, and throw away a lawn rather than naming colonies inside it. Kanamycin and chloramphenicol have their own stability. Do not assume they died on ampicillin's schedule, and do not assume they formed ampicillin's halo while they were alive.

What an enquiry needs

Name the marker gene, the drug, the host genotype, and the screen you will run after growth, because growth is not the insert. Ask for the antibiotic specification you will actually pour. A designed insert is a separate question for the custom gene synthesis enquiry reference. Proof of that insert by sequence is a question for the Sanger sequencing enquiry reference. Both are enquiry references. They do not mean EVRINTH synthesises the gene or runs the read. Put the plate and marker requirements in the quote request and ask whether a quotation is possible.