TYPES OF BLOTTING

Southern Blotting

Northern Blotting

Western Blotting
METHODS USED FOR:
 SOUTHERN BLOTTING :It is used to detect
DNA
 NORTHERN BLOTTING :It is used to detect
RNA
 WESTERN BLOTTING :It is used to detect
PROTEIN
SOUTHERN
BLOTTING
 introduction
The technique was
developed by E.M.
Southern in 1975.
The Southern blot is
used to detect the
presence of a
particular DNA
fragment in a
sample.
 PRINCIPLE
 The
principle is basically based on the
HYBRIDIZATION

 HYBRIDIZATION -: Process of forming a

double-stranded DNA molecule between a
single-stranded DNA probe and a single-
stranded target DNA
PROCEDURE CONSIST OF:-
1. DNA isolation & purification
2. Restriction digestion
3. Gel electrophoresis
4. Denaturation
5. Blotting
6. Hybridization
7. Washing
8. Autoradiography
 Step:1 DNA Isolation &
Purification
Isolation and purification of DNA from cells
-Incubate cells with detergent to promote cell lysis
-Lysis free cellular proteins and DNA
Proteins are enzymatically degraded by incubation
with proteinase
DNA is purified from solution by alcohol
precipitation
Visible DNA fibers are removed and suspended in
buffer
 Cut the DNA into different
sized fragment using
restriction endonucleases
like Eco R1
 STEP:3 Gel Electrophoresis

Technique for separation of DNA fragments

by size
Gel are solid Agarose with microscopic pores
Gel is soaked in a buffer
Stained with ethidium bromide for visualization
 Step:4&5
Denaturation
& Blotting

DNA is then denatured with an alkaline solution

such as NAOH.
DNA is then neutralized with NaCl to prevent
re-hybridization before adding the probe
Blotting is the transfer of DNA bands from
the gel to a nitrocellulose membrane
The blot is made permanent by:
Drying at 80 C or exposing to UV radiation
 Step:6 Hybridization

HYBRIDIZATION
 The labeled probe is added to the
membrane in buffer and incubated
for several hours to allow the probe
molecules to find their targets
Step:7&8 Wash & Autoradiography
 Washing
unbound probes are washed out

 For , X-ray film is

placed over the membrane
 After development, there will be
dark bands on the film wherever the
probe bound.
SUMMARY
1 .DNA is restricted with
enzymes.
2 .Extract & purify DNA
from cells.
3 .Separated by
electrophoresis.
4 .Denature DNA.
5 .Transfer to
nitrocellulose
paper(blotting).
6 .Add labeled probe for
hybridization to take
place.
7 .Wash off unbound
probe
8 .Auto radiography
 APPLICATION
 To isolated desired DNA for making of
rDNA.
 Used in prognosis of cancer and in
prenatal diagnosis of genetic disease.
 To identify specific DNA in a sample
 Used in phylogenetics analysis
 Diagnosis of HIV-1 and infection
disease
THANK YOU

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