A brief account of Southern Blotting

1. SOUTHERN BLOTTING
Saji Mariam George
Associate Professor
Assumption College Autonomous
Changanacherry

2. BLOTTING
BLOTTING – The transfer of minute amount of bio-
molecules (proteins / nucleic acids) present in bands
of electropherograms are removed from gel on to a
stable and immobilizing support such as nitrocellulose
filter, Nylon filter etc. for further investigation.
Types
1. Southern Blotting (DNA)
2. Northern Blotting (RNA)
3. Western Blotting (Proteins)
4. Eastern Blotting (Post translational modifications of
proteins)

3. SOUTHERN BLOTTING
(Edwin Southern , 1975)
• Used for screening collections of cloned DNA
fragments.
• A DNA fragment containing a specific sequence
can be identified by separating a mixture of
fragments by electrophoresis , transferring them
to nitrocellulose and hybridizing with a ³²P
labelled single stranded DNA probe
complementary to the sequence.
• The fragment containing the sequence is then
visualized by autoradiography.

4. STEPS
• DNA cleaved with a restriction enzyme.
• Restriction fragments are gel electrophoresed in an
Agarose gel.
• DNA bands in the gel are denatured into single
stranded form by alkali treatment (0.5 M NaOH).
• Transferred onto Nitrocellulose filter membrane -
 Place the gel on top of a buffer saturated filter paper
 Lay the Nitrocellulose filter membrane on the top of
the gel
 Place some dry filter paper on top of this membrane.

5. From the bottom filter paper , buffer move
by capillary action through the gel , carrying
with it the denatured DNA present in the gel .
DNA get trapped in the nitrocellulose
membrane as the buffer passes through it
because of its greater affinity → replica
pattern.
 Bake nitrocellulose membrane with ssDNA
bands at 80 ⁰ C for 2-3 hours- to fix the DNA
permanently on the membrane.

6. • Nitrocellulose membrane with the replica of
DNA bands from the Agarose gel is used for
hybridization with labelled DNA probe –
Nitrocellulose sheet is moistened with a
minimal quantity of solution containing a
³²P labelled ssDNA probe that is
complementary in sequence to the DNA of
interest.

7. The moistened Nitrocellulose is held at a
suitable renaturation temperature for several
hours to permit the probe to hybridize to its
target sequences.
Wash to remove the unbound radioactive probe.
Dry
Autoradiograph – place it over a sheet of X –ray
film – the positions of the molecules that are
complementary to the radioactive probe are
indicated by a blackening of the developed film.

9. THANK YOU