1. Restriction Digest Laboratory Restriction fragment length polymorphism

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. What is: A restriction enzyme(s)? –An endonuclease –We will focus on type II. A restriction digest?

5. Restriction Enzyme Digest

6. Examples of Restriction Enzymes http://www.accessexcellence.org/AE/AEC/CC/re_chart.php http://www.neb.com/nebecomm/tech_reference/restriction_enzymes/buffer_activity_restriction_enzymes.asp Links to restriction enzymes: http://www.neb.com/nebecomm/EnzymeFinder.asphttp://www.neb.com/nebecomm/EnzymeFinder.asp?

7. Gel Electrophoresis Following Digest

8. Analysis of Data Allows you to identify sizes of plasmid By comparing migration of digested plasmid To KNOWN SIZES of DNA.

9. Example of known sizes of DNA DNA Ladder or Markers

10. A map gives the size of fragments A map gives the number and position of cutting sites JUST AN EXAMPLE Not your map! Plasmid map 1500 800 60 600 1400

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

12. 2 cutting sites 2 fragments 2 cutting sites 3 fragments Plasmid DNA Linear DNA

13. Today’s experiment Restriction of Digest of plasmid DNA using two restriction enzymes.

14. Please refer to page 10 of the handout (6 groups) Each Group set up a rack with: –Reaction buffer –water –Plasmid DNA –Ava I –SacII –Loading Dye –Standard (marker or ladder) DNA Label four microfuge tubes 1→4 Must keep on ice

15. Pipette the samples as shown on page in handout—not lab manual.

16. 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

17. 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

19. Gel Electrophoresis Load 25 ul per well Run gel at 75 volts until the dye front is approximately half-way down gel. Take photograph