3. Southern
blotting is a
process that transfer the DNA
fragments that separated by
electrophoresis into a
nitrocellulose membrane/
nylon.

4. To
determine the DNA sequence
between 2 DNA samples .
Whether the DNA sequence is
present or not.
There
is also lot of blotting
methods like
nothern,southern,western blotting.

5.  Southern
blotting involved in five main
processes
1.digest DNA
2.RUN digest DNA into agarose gel
3.Denature the DNA
4.Transfer the denatured DNA to
the membrane
5. hybridization.
6.visualization

6. 1. DNA DIGEST
Digest the SAMPLE BY USING specific
Restriction enzymes .
2.TRANSFER
DNA digest into
agarose gel
3.denature of DNA
separate double-stranded DNA
into single-stranded DNA. Only
ssDNA can transfer.(0.5M NAOH,0.2M HCL)

8.  Nitrocellulose
membrane or nylon can be
used for this step. because they have small
pores that allows the capillary motion.
 Transfer is usually done by capillary force
 CAN use a vacuum blot apparatus instead of
capillary action

10.  AFTER
THAT treat the membrane with UV
light or bake the sample in 800c. This causes
cross links the DNA to the membrane. (via
non-covalent semi permanent bonds)
 Now the DNA became immobilized in
membrane.

12.  We
need a radio active probe to determine
presence of a specific sequence in the DNA
FRAGMENT.
 The blotted DNA fragments then dipped into
a buffer solution and mix with radio active
probes and sealed and rotated..

13.  We
want to make sure there is enough probes
to bind with DNA fragments.
 In autoradiograph
THEY ENCOUNTERED A PROBLEM !!!!
the probes that didn’t bound with DNA
fragments also there. this effect our final
result.(probes bounded with membrane)
So they introduced a technique call
prehybridization.

14.  This
step involved in closing the binding site
of menbrane other than DNA fragments.
 For this a special prehybridization solution is
used.
 Such as-1.polyvinylpyrrolidone
2.bovine serum albumin
3.dried milk
4.other organism DNA (salmon sperm DNA)
After this, membrane is washed and ready
for hybridization.

15.  Visualize
your radioactively labeled target
sequence BY autoradiograph

16.  Using
nylon instead of nitrocellulose
membrane is speed up the movements of
DNA.(membrane=18 h,nylon=2h)
 Electro blotting –using electrophorosis
between gel to nylon speed up the DNA
movements.
 Vacum blotting