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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. done. Restriction Digest of DNA Electrophoresis

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southern blot
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 blot3
Southern Blot

done

  • Restriction Digest of DNA
  • Electrophoresis
  • Denaturation/Depurination
  • Blotting Step
  • Probing

To do

To do

To do

2 wks

slide5

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
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 volume add 40 u l per well must warm 5 min at 65c
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
Depurination Step
  • 8 minute (maximum) incubation in 100 ml .25N HCl
  • Rinse gel several times with 100 ml distilled water

depurination

denaturation step
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
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
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
Setting up the Southern Blot transfer
  • Plastic wrap
  • Inverted Gel
  • Nylon Membrane
  • Filter paper
  • Paper towels
  • Weight
  • Overnight incubation at room temperature
analysis of southern blot

Analysis of Southern Blot

A non-isotopic method of detection

remember the southern blot requires
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
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
You will probe the blot today
  • Be sure and refer to your manual during these steps!
you will probe the blot today24
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
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
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
slide27

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
Agarose gel electrophoresis
  • Biotinylated DNA fragments labelled probes
  • Avidin labelled enzyme
  • Avidin has 4 binding sites for biotin
slide30
Substrate: 5-Bromo-4-chloro-3 indolyl phosphpate (BCIP)Product: Nitro Blue Tetrazolium to be reduced to an insoluble visible product.