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 done • Restriction Digest of DNA • Electrophoresis • Denaturation/Depurination • Blotting Step • Probing To do To do To do 2 wks
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 125 volts for 45-60 minutes • Lecture will be given while samples electrophorese.
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
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 10-15 min. • Replace with fresh denaturation solution for 10-15 minutes
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 for similarities between child and parents.
Analysis of Southern Blot A non-isotopic method of detection
Remember the Southern Blot requires… • that genomic DNA be first “digested” into smaller fragments • that the DNA be separated on a gel • that the DNA be denatured into single stranded DNA
Southern Blot requires that… • you “probe” the fragments with a complementary sequence of DNA or RNA • you have a means to “visualize” the binding between the probe and the DNA
You will probe the blot today • Be sure and refer to your manual during these steps!
You will probe the blot today • First you must prepare the nytran membrane • Use warm blocking buffer 45 minutes • Remove blocking buffer • Rinse container with water • Add probe and mix well • Incubate 10 minutes
Next: many rinsing steps • Rinse with 400 mls detection buffer for 10 min • Rinse again with 200 mls for 15 min • Rinse again with 200 mls for 15 min • Be sure and agitate the solution with the buffer for complete rinsing
Color Development • I will prepare the substrate • Add 8 mls of substrate • Place DNA face down • Place in 37C water bath • Color should develop within 15-20 min
Let’s look at some animations and examples of Southern Blots used in actual cases: http://vector.cshl.org/resources/dnadetective.html http://vector.cshl.org/resources/BiologyAnimationLibrary.htm
Agarose gel electrophoresis • Biotinylated DNA fragments labelled probes • Avidin labelled enzyme • Avidin has 4 binding sites for biotin
Substrate: 5-Bromo-4-chloro-3 indolyl phosphpate (BCIP)Product: Nitro Blue Tetrazolium to be reduced to an insoluble visible product.