Explainers

Short, cited explanations of what is on a plasmid map, each linked to a real plasmid and to the stop of the cell tour it illustrates.

• How to read a plasmid map

  Origin, resistance marker, promoter, cloning site and insert, explained on a real plasmid and linked to the structures inside the cell.

• The origin of replication and plasmid copy number

  How a ColE1-type origin starts replication with an RNA primer, why RNA I sets the copy number, and why pUC plasmids are copied more often than pBR322.

• Ampicillin resistance: the bla gene and β-lactamase

  What the bla gene on a plasmid map encodes, how β-lactamase protects a cell from ampicillin, and how it is used to select cells carrying the plasmid.

• Promoters on a plasmid map

  What a promoter is, how the lac promoter and operator are regulated, and why expression plasmids use the T7 promoter.

• Coding sequences and reading frames

  What a CDS arrow on a plasmid map means, how a reading frame is read in triplets from a start codon to a stop, and how to open the protein it encodes.

• Restriction enzymes and their sites

  How restriction enzymes recognise short DNA sequences and cut them, what sticky and blunt ends are, and how Type IIS enzymes cut outside their sites.

• dam and dcm methylation and blocked sites

  Why some restriction sites do not cut plasmid DNA grown in E. coli, what the dam and dcm methylases mark, and what "may be blocked" means.

• Choosing a diagnostic restriction digest

  How to choose a digest whose bands tell a correct construct from one with the insert missing or reversed, and how to read a schematic gel.

• Sequencing primers and where they bind

  How Sanger sequencing reads a plasmid from a primer, which universal primers bind common vectors, and what a one-mismatch site means.

• Selection markers beyond ampicillin

  Tetracycline, kanamycin and other resistance genes on plasmid maps: what each marker lets you select, and why pBR322 carries two.