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 To do
Denaturation/DepurinationTo do
Blotting Step To do
Probing
2 wks
done
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.
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
Depurination Step
• 8 minute (maximum) incubation in
depurination
100 ml .25N HCl
• Rinse gel several times with 100 ml
distilled water
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
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
for similarities between child and parents.
Southern Blotting
DNA Fingerprinting
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/BiologyAnimationLibrar
Agarose gel electrophoresis
• Biotinylated DNA fragments labelled
probes
• Avidin labelled enzyme
• Avidin has 4 binding sites for biotin
Biotin-DNA binds to avidin labelled
alkaline phosphatase.
Substrate: 5-Bromo-4-chloro-3
indolyl phosphpate (BCIP)
Product: Nitro Blue Tetrazolium to be
reduced to an insoluble visible
product.
Southern Blot Analysis