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&. 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. - PowerPoint PPT Presentation
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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
1. Restriction enzymes “cut up” the DNA samples into fragments
2. DNA samples placed into wells at one end of the chamber
3. 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?
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