Southern Blotting DNA Fingerprinting

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

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

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

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.

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. During your electrophoresis we will continue our discussion of Genomics.

How to handle your gel after electrophoresis…

Load Gels and then we will discuss recombinant DNA technology

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

.25N HCL NaOH/NaCl Depurination Denaturation

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

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

General Schematic for Southern Blot: DNA

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

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

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

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

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