ISOLATION OF
GENOMIC DNA

WHAT IS DNA?
• Genetic material of an organism
• Polymer of Deoxyribonucleotides
• Found in Nucleus, Mitochondria, and Chloroplast
• Consist of four types of bases:
Nitrogen
bases
Purine
Adenine
(A)
Guanine
(G)
Pyrimidine
Cytosine
(C)
Thymine
(T)

WHAT IS THE STRUCTURE OF DNA?
• Primary structure of DNA Secondary structure of DNA Tertiary structure of DNA

WHY DO WE NEED TO ISOLATE
DNA?
SIGNIFICANCE
OF ISOLATION
OF DNA
FORENSIC
STUDY
GENETICS
STUDY OF
DISEASES
DETECTIN
G
MICROBES
IN
ENVIRONM
ENT
GENOME
SEQUENCI
NG
PATERNITY
DETERMIN
ATION
DIAGNOSIS
DRUG
DEVELOPM
ENT

WHY DO WE NEED DIFFERENT
PROTOCOLS FOR DNA ISOLATION?
• Due to differences in the cell structure of organisms, we require different protocols for
the isolation of DNA.
• For example, the CTAB method is used for the isolation of DNA from plants and fungi
due to the presence of cellulose and chitin cell walls respectively
• In comparison to plants and fungi, isolation from animal cells requires lesser effort and
different approaches to isolate DNA as animal cells are not enveloped in a cell wall.

STEPS FOR ISOLATION OF
DNA

Weigh mice
liver tissue
200gm

Extraction
buffer
(2.5mL)
Mice liver
tissue 200gm
Add 200µL of 10% SDS
Help to break the
tissue to release
DNA content
Breaks down cell
membranes and alters the
structure of proteins
Chemical
s
Stock Working Volume
(mL)
EDTA 0.5M 0.1M 10
SDS 10% 1% 5
Milli-Q
water
35

Add twice volume of 2M
NaCl solution
Shake vigorously for 2
minutes
Alters the structure of some cell
proteins. As a result, proteins get
denatured and placed at bottom of the
tube
Add 10µL Proteinase K
and 5µL RNase A
Incubate this cell lysate at 50ºC for
30minutes

Centrifuge lysate at 10,000rpm for
10minutes at 4ºC
Transfer supernatant and discard the
pellet
Transfer 500µL of supernatant
into 1.5mL tubes

Add an equal volume of Chloroform:
Isoamyl alcohol (24:1) and gently
mix it
Centrifuge lysate at 10,000rpm for
10minutes at 4ºC
Carefully transfer aqueous
layer into fresh tube

Add an equal volume of Absolute
Alcohol and gently mix it
Centrifuge lysate at 10,000rpm for
10minutes at 4ºC
Decant the supernatant

Add 70% Ethanol for washing
Centrifuge lysate at 10,000rpm for
10minutes at 4ºC
Decant the supernatant and
dry pellet properly

Store in Milli-Q water
Check at 0.8% Agarose Gel

THANK YOU