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

10. You must carefully follow page 3-65 6 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. Obtain a rack and: ●1. Obtain new microfuge tubes and label 2-8

12. 2. Place these tubes also on your rack  Tube L= Ladder “known sizes of DNA”  Tube P=Plasmid DNA “cocktail”  Tube A: AfaI  Tube B: Mae I  Tube C: Xma I  Tube D: Loading Dye  Tube W: Water note these enzymes are different than your lab manual

13. Place tubes … On ICE

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

15. 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

16. Restriction Digest AFTER 1 hour DIGESTION: You must add 5 ul 10X loading dye to your samples (not to the ladder (L)). Pre-heat all samples including ladder for 3-5 min. at 65C

18. Gel Electrophoresis Load 25 ul per well Run gel at 75 volts for 45 minutes Take photograph