Southern Blotting DNA Fingerprinting

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 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 • Each group should load their gel • Run gel at approximately 75 volts for approximately 20-25 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 VolumeAdd 40 ul per wellMUST 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.