1. Southern Blotting DNA Fingerprinting

2. Southern Blot A Southern Blot identifies specific sequences of DNA
A Southern Blot may be used to determine a DNA fingerprint
A Southern Blot may be used in forsenic medicine

3. Southern Blot Restriction Digest of DNA Electrophoresis
done
Restriction Digest of DNA
Electrophoresis
Denaturation/Depurination
Blotting Step
Probing
To do
To do
To do
1wk

4. Today’s laboratory: DNA fingerprinting in a hypothetical paternity determination.

5. A Paternity
Case
In this hypothetical case, DNA was extracted from samples obtained from two possible fathers, a mother and child. The DNA was were cleaved with a restriction enzyme.

6. Loading Gels
This part of the lab will be started for the class prior to the beginning of the lab.
Run gel at approximately 75 volts for approximately minutes or to when the dye-front has moved NOT more than half-way down the gel.

7. How to handle your gel after electrophoresis…

8. Load Gels and then we will discuss recombinant DNA technology

9. Sample Volume Add 40 ul per well MUST WARM 5 min. at 65C
Samples
A. Standard DNA fragments
B. Mother DNA cut with restriction enzyme
C. Child DNA cut with restriction enzyme
D. Possible Father #1 DNA with restriction enzyme
E. Possible Father #2 DNA cut with restriction enzyme

10. .25N HCL
NaOH/NaCl
Depurination
Denaturation

11. Depurination Step 8 minute (maximum) incubation in 100 ml .25N HCl
Rinse gel several times with 100 ml distilled water
depurination

12. Denaturation Step After rinsing gel with water
Add 100 ml DNA denaturation solution 5-10 min.
Replace with fresh denaturation solution for 5-10 minutes

13. General Schematic for Southern Blot: DNA

14. Setting up the Southern Blot: pg. 3-82
Line a tray with plastic wrap
Place “denatured” gel upside down on wrap
Pre-wet nylon membrane in denaturation solution for 2-5 minutes
Place nylon on top of inverted gel

15. Setting up Southern Blot: pg. 3-82
Place filter paper on top of nylon membrane
Remove air bubbles
Place stack of paper towels on top of filter paper
Place empty 400 ml beaker on towel
Incubate overnight

16. Setting up the Southern Blot transfer
Plastic wrap
Inverted Gel
Nylon Membrane
Filter paper
Paper towels
Weight
Overnight incubation at room temperature

17. Overnight the ssDNA will diffuse by capillary transfer from gel onto nylon membrane!

18. Next week we will stain this membrane and look for similarities between child and parents.