DNA ISOLATION
Aim
To isolate the DNA from a given fish tissue sample.
Principle
The isolation of DNA usually begins with the lysis of cells or tissues in order to destroy
the protein structures and allows the release of nucleic acids from the nucleus. Lysis is
carried out in a lysis solution containing important ingredients: sodium chloride which
provides an osmotic shock to the cells; Tris.HCl, which is a buffer to retain constant pH;
EDTA, which sequesters the divalent metal ions that is required for nuclease activity and
thereby inhibiting its action; a detergent, usually SDS, which disrupts the cell membrane
and nuclear envelope, causing the cells to burst open and release their DNA. The DNA is
still rapped very tightly around histone proteins. Proteinase K (a serine protease) is the
common enzyme used in DNA extraction that cuts apart the histones to free the DNA and
finally results in the breakdown of cells and dissolving of membranes.
The nucleic acids are then purified from the protein- nucleic acid complex by phase
extraction with a mixture of organic solvents namely Phenol, Chloroform and Isoamyl
alcohol in a ratio of 25:24:1. Phenol dissociates proteins from DNA. Chloroform denatures
the proteins and lipids and helps to maintain the separation of the organic and aqueous phase.
It also makes the DNA less soluble in the phenol, thus reducing losses to the organic phase.
Isoamyl alcohol is often added to prevent foaming. At pH 7-8, the DNA partitions to the
aqueous phase while the protein is denatured and extracted into water- immiscible organic
phase, which is separated from the nucleic acid containing aqueous phase by centrifugation.
When large amount of protein is present, it forms a white precipitate between the organic
and aqueous phase.
The DNA is then precipitated with cold ethanol or isopropanol after adjustment with 3M
sodium acetate and then centrifuging. The DNA is insoluble in the alcohol and will come
out of solution, and the alcohol serves as a wash to remove the residual salts. The alcohol is
then removed, and DNA is stored in a biological buffer, like TE (Tris-EDTA) buffer.
Contaminating RNA in the DNA sample can eliminated by digestion with an RNase.
Materials required
• Fish tissue
• Forceps
• Mortar and pestle
• Centrifuge
• Vials
• Micro pipette
• Pipette tips
• Water bath
• Filter paper
• Blotting paper
Reagents 1. Extraction buffer
• 1M Tris HCl (pH 8.0)
• 2.0.5M EDTA (pH 8.0)
• 3.2% Triton X-100
• 4.1% SDS
• 5. 1 M NaCl
2. TE Buffer
• Tris base – 10mM
• EDTA – 1mM
3. Phenol: Chloroform : Isoamyl alcohol – 50:48:2
4. 70%, 100% ethanol ( Ice-cold)
5. Sodium Acetate – 3M
Procedure
20-100 mg of fish tissue was taken using a sterile scalps and forceps
The tissue was homogenized in 1ml of extraction buffer using a sterile mortar and
pestle The paste was collected into 1.5ml micro centrifuge tube and incubated at
55-650C for 1 hour in a water bath.
It was centrifuged at 5000rpm of 10 minutes
Supernatant was removed and taken a new microcentrifuge tubes
Equal volume of Phenol: Chloroform: Isoamyl alcohol ( 25:24:1) was added and
mixed slowly and thoroughly by repeated inversion of micro centrifuge tube.
It was centrifuged at 12000rpm for 10 minutes.
The top aqueous layer was collected in a fresh micro centrifuge tube without
disturbing the intermediate layer
Chloroform : Isoamyl alcohol in the ratio of 24:1 was added and mixed slowly and
thoroughly by inversion of the tube
It was centrifuged again at 12000 rpm for 10 minutes
The aqueous layer was collected into a fresh micro centrifuge tube and 0.1 volume of
3M Sodium Acetate and equal volume of ice cold ethanol (100%) were added. The
solution was mixed thoroughly until the DNA pellet is obtained
It was then incubated at -200C for 10 minutes and centrifuged at 10000rpm for 10
minutes. The supernatant ( ethanol) was decanted
The pellet was washed with 70% Ethanol by centrifuging at 1000rpm for 10 minutes
and the Ethanol was carefully decanted without loss in the pellet.
DNA pellet was air dry at room temperature by keeping the tube opened and also
blotted using filter paper / blotting paper
The pellet was re -suspended in 50-100 µl of TE buffer or sterile triple distilled water.
Result
The DNA was isolated from the given fish tissue sample and confirmed in
Agarose gel electrophoresis.
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