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)? A restriction digest?
An endonuclease
We will focus on type II.
A restriction digest?

5. Restriction Enzyme Digest

6. Examples of Restriction Enzymes
Links to restriction enzymes:

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 60
800
600
JUST AN EXAMPLE
Not your map!
1500
Plasmid map
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. Linear DNA
Plasmid DNA
2 cutting sites
2 fragments
2 cutting sites
3 fragments

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
NotI for AM lab
SfiI for AM lab Or
AluI for PM lab
HphI for PM lab
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