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Ex - No. Genomic DNA Isolation From Fish Fins Using Phenol-Chloroform Method

This document describes a phenol-chloroform method for isolating genomic DNA from fish fins. The method involves lysing cells from fin tissue, treating with proteinase K to release DNA from histones, and then purifying the DNA through multiple steps of phase separation using phenol, chloroform and isoamyl alcohol to separate DNA in the aqueous phase from proteins in the organic phase. The DNA is then precipitated with ethanol, washed, dried and resuspended in TE buffer for storage.

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70 views2 pages

Ex - No. Genomic DNA Isolation From Fish Fins Using Phenol-Chloroform Method

This document describes a phenol-chloroform method for isolating genomic DNA from fish fins. The method involves lysing cells from fin tissue, treating with proteinase K to release DNA from histones, and then purifying the DNA through multiple steps of phase separation using phenol, chloroform and isoamyl alcohol to separate DNA in the aqueous phase from proteins in the organic phase. The DNA is then precipitated with ethanol, washed, dried and resuspended in TE buffer for storage.

Uploaded by

mercyaugustin
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Ex.No.

Genomic DNA isolation from fish fins using Phenol-Chloroform


method

Principle
DNA isolation refers to the process of extracting DNA from a cell in a pure form. The
extraction of DNA is an important preliminary step in which purified DNA is obtained from
other cellular components such as proteins, RNA and lipids. DNA can be isolated from any
nucleated cell from diverse sources, both living and dead, such as whole blood, hair, sperm,
bones, nails, tissues, faeces, shed feathers, egg shells, saliva, epithelial cells,
urine, bacteria, animal tissues or plants.
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. 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.

Reagents required

 Lysis Buffer (TEN Buffer)


o 50mM Tris
o 10mM EDTA
o 100mM NaCl
 10% SDS
 Proteinase K (10mg/ml)
 Tris-saturated Phenol
 Phenol: Chloroform: Isoamyl alcohol (25:24:1)
 Chloroform: Isoamyl alcohol (24:1)
 Isopropanol
 70% ethanol
 10x TE Buffer
o 10mM Tris
o 1mM EDTA

Procedure
 Take 50-100mg of fin tissue in a 2.0 ml eppendorf tube
 Homogenize in 500μl of TEN buffer using sterile scissors/homogenizer
 Add 50μl of 10% SDS & 5μl of Proteinase K
 Incubate at 56 deg for overnight in a water bath
 Add equal volume (555μl) of tris-saturated phenol and mix properly
 Centrifuge at 10,000 rpm for 10 minutes at 4deg
 Transfer the aqueous phase to a fresh 1.5 ml eppendorf tube
 Add equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) and mix slowly
and thoroughly by repeated inversion of tube
 Centrifuge at 10,000 rpm for 10 minutes at 4deg
 Transfer the aqueous phase to a new 1.5 ml eppendorf tube
 Add equal volume of Chloroform: Isoamyl alcohol (24:1) and mix slowly and
thoroughly by repeated inversion of tube
 Centrifuge at 10,000 rpm for 10 minutes at 4deg
 Transfer the aqueous phase to a new 1.5 ml eppendorf tube
 Add 0.6 volume of isopropanol and mix thoroughly
 Centrifuge at 12,000 rpm for 10 minutes at 4deg
 Discard supernatant and wash the pellet with 500μl of 70% chilled ethanol
 Centrifuge at 12,000 rpm f0r 10 minutes and discard the supernatant
 Air dry the DNA pellets at room temperature by keeping the tube opened and also
blot using filter paper
 Resuspend the pellet in 50μl 1x TE Buffer and store at -20 deg
 Check the quality of Genomic DNA on an agarose gel.

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