Southern Blotting

General Scheme for Southern Blot

Restriction Digest
Gel Electrophoresis
DNA Preparation:
Denaturation/Depurination

Transfer to filter: Blotting

Detecting DNA: Probing

Southern Blot Analysis
Southern Blot
• Restriction Digest of DNAdone

• Electrophoresis
• Denaturation/Depurination
• Blotting Step (overnight)
• Detection of DNA (next lab)
Format for today’s laboratory:
• 5 groups

• Each group
– loads and runs agarose gel
– Completes depurination/denaturation steps
– Sets up DNA transfer
 DNA Fingerprinting Analysis
40 ul sample per well
Sample Tube
Label
Standard DNA fragments A
Mother DNA cut with Enzyme B
Child DNA cut with Enzyme C
Possible Father #1 DNA cut D
with Enzyme
Possible Father #2 DNA cut E
with Enzyme
 Depurination and Denaturation Step
page 3-83 with changes

• Place gel in 100 ml 0.25N HCl for a maximum

of 8 minutes.
• Rinse gel several times with 100 ml distilled
water
• Place gel in 100 ml DNA denaturation solution
for 15 min. with occasional shaking.
• Replace with 100 ml fresh denaturation solution
for 15 minutes
 The reason for depurination
• Depurination: 0.25 N HCl
(used to help in a teaching lab)

– Reduces binding between DNA strands by

creating apurinic sites (loss of purine base)
– Predisposes the DNA-phosphate backbone
to cleavage by alkaline solution
– Smaller fragments transfer better
 The reason for denaturation:
• Denaturation:NaOH/NaCl

– Double-stranded DNA is converted to

Single-stranded DNA
– Sugar-phosphate backbone is cleaved at
the sites of depurination
– This step must always be performed.
 Setting up the Southern Blot Transfer
page 3-84

• Line a tray with plastic wrap

• Place “denatured” gel upside down on wrap

• Pre-wet nylon membrane in denaturation

solution for 5 minutes

• Place nylon on top of inverted gel

Setting up the Southern Blot Transfer
Page 3-84
• 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
Order of Components from the bottom:
Gel
Membrane
Filter paper
Paper towels
Weight
 Overnight Incubation at Room Temperature
Single stranded DNA will diffuse by capillary
transfer from gel to nylon membrane
 Analysis of Southern Blot

A Non-isotopic Method of DNA Detection

Revised from Lab Manual Instructions
 Comparing Our Experimental System
to Southern Blots in Research Labs
In research lab In teaching lab
Radioisotopic Method Non-Isotopic method
DNA Restriction Restriction
Fragments Fragments Fragments

Probe Radioactively-labeled None

DNA or RNA
Visualization Radioactivity from DNA Stain allows
of DNA probe exposes X-ray shows all fragments
Fragments film, subset of on gel
fragments observed
 Non-isotopic Detection of DNA
These directions REPLACE those
found on 3-86  3-89
• Place the membrane with the DNA side up in
100 ml of Blue-Blot solution.
• Soak the membrane at room temperature for
10-15 minutes.
• Remove the membrane with forceps and rinse
in 200 ml distilled water.
• Replenish the distilled water 3-4 times or until
the membrane is destained and DNA bands
are clearly visible.