1. &
Gel
Plasmid
Electrophoresis
Mapping

2. 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

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

4. 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

6. 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)

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

8. 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

9. Example

10. 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

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

12. 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

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

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

15. Which is the correct model?