1. Restriction Digest Laboratory

2. Reminder You have transformed bacteria with plasmid DNA
You have isolated plasmid DNA
Today you will perform an RFLP analysis
& Confirm your Plasmid Isolation

3. This is the third and final section of your lab report.
Digest plasmid DNA
Determine number of cutting sites
Determine location of cutting sites
Determine size of fragments
Present the “map” of the plasmid in your report
The steps in BLUE you will complete outside of class as part of your data analysis.

4. Restriction Enzyme Digest

5. DNA Separation following Digest

6. Markers: Size Identification

7. RFLP provides a map of your plasmid
A map gives the number and position of cutting sites
A maps gives the size of fragments

8. Remember Plasmid is Circular
Circular DNA: the number of fragments=number (N) of cutting sites
versus
Linear DNA: number of fragments=N+1

9. Linear DNA
Plasmid DNA
2 cutting sites
2 fragments
2 cutting sites
3 fragments

10. You must carefully follow page 3-65
7 groups for today’s experiment
Each group should set up a rack with the tubes necessary for the restriction digest
Assign a member of your group to pick up sample tubes.

11. Place tubes …
On ICE

12. Pipette the samples as shown on page 3-65

13. After you are finished pipetting your samples
Place samples at 37C for 1 hour
After 1 hour you will be ready to load your gel

14. AFTER 1 hour DIGESTION: You must add 5 ul 10X loading dye to your samples (not to the ladder (L)).

15. Gel Electrophoresis Load 20 ul per well
Run gel at 75 volts for 45 minutes
Take photograph