& Gel Plasmid Electrophoresis Mapping

Plasmid Mapping Purpose: identifying the position of “restriction sites” on a fragment of DNA Can help identify the position of a specific gene within DNA Restriction enzymes (endonuclease) cut a plasmid into smaller fragments of DNA

Gel Electrophoresis Procedure used to separate the DNA fragments by their size

Gel Electrophoresis Restriction enzymes “cut up” the DNA samples into fragments DNA samples placed into wells at one end of the chamber An electric current is applied to the gel – smaller fragments move towards the positive end faster than larger fragments

Example This fragment of DNA is 7.0 kb (kilobases) in length When digested with Hind III enzyme, two fragments result (a 6.2 kb fragment and a 0.8 kb fragment)

Example Thus, we know there is a Hind III restriction site 0.8 kb from one end of the fragment

Example If we digest the fragment with another enzyme, Sal I, two fragments result Now, they are 5.8 kb and 1.2 kb in length

Example

Example If the restriction sites for both enzymes are on the same end, we’d expect the 0.8 kb fragment to be within the 1.2 kb fragment

Example A double-enzyme digestion would give three fragments (0.4, 0.8, and 5.8 kb)

Example However, if the restriction sites are at opposite ends, we’d expect the 0.8 kb fragment to be within the 5.8 kb fragment

Example A double-enzyme digestion would give three fragments (0.8, 5.0, and 1.2 kb)

Example In order to determine which map is correct, we must digest the DNA with both enzymes

Which is the correct model?