A glossary of cloning terms

A cloning plan is ready when each word points at ink on the map. Vector, insert, backbone, MCS, overhang, blunt, sticky, ligase, phosphatase, transformation, and clone are the words that cause silent swaps. You are deciding whether this plan can be pipetted or whether it still uses two names for one feature and no name for the junction. This is a decision guide, not a thin dictionary and not a kit insert.

The bench path that uses these words is plasmid cloning from insert to colony. How compatible ends are joined is in restriction ligation and Gibson assembly. Here you only mark the plan.

Put the file where the words can land

Open the annotated map, a GenBank-style record or the equivalent your group trusts, beside the written plan. NCBI GenBank is one public place a reference record lives. Your plasmid may only exist as a local file. Either way the file has to show bases and features. A gene nickname on a slide is not a map.

Read the plan once and underline every term in the list above. Then mark it on the file. If you cannot mark it, the plan is using a word it has not defined. Stop and redraw before you order an enzyme or thaw competent cells.

What each word is on the molecule

The vector is the DNA molecule that carries an origin and a selectable marker and is meant to accept the insert. Mark the whole molecule you will cut or assemble into. Say in the filename whether the insert is already present. Calling both the empty plasmid and the finished construct "the vector" is how the wrong tube gets transformed.

The backbone is what remains when the insert is removed from that file: origin, marker, and any promoter or tag you did not add as this week's fragment. Mark it as an arc. Copy number, a leaky promoter, and a leftover counterselection gene are backbone facts. They are not properties of the insert's name.

The insert is the span you intend to add, with ends and orientation. Mark the first and last base, and an arrow for direction relative to any promoter you will use. A gene symbol is not an insert until the bases are the ones you mean. PCR, a restriction fragment, and a synthetic piece are all inserts. They are not interchangeable sources, but they occupy the same mark on the map.

The MCS, the multiple cloning site, is a short cluster of restriction sites, often sitting in a lacZ alpha fragment so colour screening is possible. Mark those bases. Then search the rest of the backbone for the same site. An MCS is a convenience, not a promise that the site is unique. If your plan never cuts there, write "MCS unused" so nobody treats a colourless colony as evidence.

An overhang is a single-stranded stretch past the duplex. Write its sequence and whether it is a 5-prime or a 3-prime extension. Restriction enzymes and designed oligos both create overhangs. The mark is the sequence, not the word sticky.

Blunt means both strands end at the same base. There is nothing for a partner to base-pair with before ligase acts. Blunt joins do not enforce orientation. If the plan says directional and the ends are blunt, the plan contradicts itself. Mark that contradiction. Do not start the digest hoping the word directional will create an overhang.

Sticky describes two ends whose overhangs can base-pair. It is a relationship. One end is not sticky in the abstract. Mark both partners. If the overhangs differ by a base, they are incompatible, even though each was produced by a real enzyme.

Ligase is the enzyme class that seals a nick when a 5-prime phosphate sits next to a 3-prime hydroxyl. T4 DNA ligase is the usual class. It needs ATP. Some assembly mixes include a ligase activity among other activities. Mark which class the plan calls for. "We will join it" is not a mark.

Phosphatase removes 5-prime phosphates. Used on the vector, it makes self-ligation harder, because the vector ends cannot be sealed to each other. The insert must still bring phosphates. Mark the tube that is treated. Phosphatase on both partners leaves ligase with nothing to join. If the plan says "dephosphorylate" and does not name a tube, you do not yet have a plan.

Transformation is the introduction of the plasmid into a competent cell. Mark the strain and the method class, chemical or electroporation. It is not a synonym for ligation, and it is not a colony. The antibiotic on the plate is part of this mark, because it has to match the marker on the backbone.

A clone, in this glossary, is a colony whose plasmid has passed the checks the plan listed. A resistant colony is a candidate. Colony screening can show size or a junction band. Sequence is the record when the insert was amplified or synthesised, and whenever orientation could still be wrong. Do not let the plan call the ligation tube a clone. There is no colony in that tube.

Where a plan fails while it still sounds fluent

The insert arrow is missing, so orientation is a hope. Colony screening with primers that do not ask about direction will bless both orientations. Add one mark that would fail if the fragment were backwards, or name the junction read. The Sanger sequencing enquiry reference is a way to specify that read later. It does not replace the arrow on the map today.

The MCS is cited for a site that also sits in the backbone. The digest will cut twice. The gel will not match the cartoon. Search the file before you trust the site list printed on a supplier map from a different version of the plasmid.

Ligase is named and phosphatase is named on both fragments. That plan cannot produce a sealed circle. Move the phosphatase mark to the vector, or delete it if the two ends are already incompatible and self-ligation is unlikely. Do not stack both treatments to feel careful.

Transformation is planned into a strain the backbone cannot survive, for example a counterselection cassette in a sensitive host, or a methylated site the enzyme card says will not cut. The glossary mark for transformation includes the strain. A strain line left blank is an incomplete plan.

The word clone appears in the first sentence, before any check. Rewrite it as candidate. Archive language can wait until the sequence file exists.

Safety and other people's plans

Recombinant DNA follows your institutional rules. Marking a map is not approval to build the insert. The WHO Laboratory biosafety manual, 4th edition is background for containment thinking. A hazardous open reading frame needs the committee's decision written somewhere the plan can point to. Do not hide that pointer in a personal notebook while the map looks ordinary.

Names that have to travel

A plan full of local nicknames fails when a second site has to order the enzyme or repeat the strain. Write the enzyme class, the recognition sequence, the antibiotic, and the strain genotype in words a stranger can search. That is specification writing, not decoration. The molecular biology catalogue can be asked for an enzyme class only after the map says which class. Put the marked file with the quote request. If the insert is a designed sequence rather than a fragment you already hold, the custom gene synthesis enquiry reference is a prompt for that specification. Ask whether a quotation is possible. Send the map with the terms marked, not a paragraph of gene names.