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PRACTICAL 1: ISOLATION OF DNA FROM

RAT LIVER

Aim: To isolate DNA from rat liver


Reagents
1. 0.1M-NaCl, 0.1M EDTA, 1% (w/v) Sodium dodecylsulphate (pH 8.0) [Solution A]
2. Chloroform-isoamyl alcohol (24:1, v/v)
3. Ethanol (96%)
4. 0.15M NaCl, 0.01M Sodium citrate solution containing ribonuclease (10g/ml)
[Solution B]
5. Sodium dodecylsulphate (5% w/v) in water
6. Chloroform
7. 0.15M NaCl, 0.01M Sodium citrate saturated phenol.
8. 0.15M NaCl, 0.015M Sodium citrate

The DNA collected was dissolved in 15ml of solution B, in a boiling tube.


Solution B contains ribonuclease which is an enzyme used to break down
RNA so that contaminating RNA is removed. The tube was incubated at
37C for 30 minutes, so that the RNase is able to work at optimal
temperature. After 30 minutes, a solution of 5% (w/v) sodium dodecyl
sulphate (3ml) was added so that the final concentration is 1% (w/v).
The sodium dodecyl sulphate eliminates and precipitates RNA.

The solution was transferred into a 250ml conical flask and extracted
(Shaking with mechanical shaker for 30 minutes) with an equal volume of
chloroform. Chloroform plays a role in denaturing proteins and dissolving
lipids. The solution was then centrifuged at 2500 r.p.m. for 5 minutes.

The supernatant was removed and transferred into a 250ml conical flask.
To the supernatant, an equal volume of solution C was added. Solution C
contains phenol which is a strong denaturing agent for proteins, the
contaminants aggregate into the organic phenol phase and the DNA
remains in the supernatant. The solution was put into the shaker for 20
minutes and then centrifuged at 2500 r.p.m. for 15 minutes.

The supernatant which contains the DNA was removed and transferred
into a beaker. One volume of cold ethanol is added to the supernatant
with stirring. The ethanol dehydrates the DNA so that it is easily
removed from the solution. The cold temperature prevents the DNA
from breaking apart. The DNA was collected on the glass rod and rinsed
with 3ml 96% ethanol.

The spooled DNA was dissolved in SSC. SSC is used as a hybridisation


buffer to control stringency during the washing step. It also increases
ionic strength so that DNA precipitation increases.

A sample of 5g of fresh rat liver was taken into a weighing boat.


This liver was extracted from rats which have ingested only water
24 hours prior to the extraction. The rats were not fed food, since
the sample would become glycogen contaminated.

The liver sample was run under tap water to remove the blood, and
was blotted dry with filter paper to remove excess water. The liver
was transferred to a mortar where it was minced to a paste form
using scissors, and then pulped with a pestle. This pulp was then
transferred into a 200ml beaker.

To the liver tissue in the beaker, 8 volumes (roughly 40ml) of solution


A was added. The 0.1M NaCl serves as an osmotic stabilizer. The 0.1M
EDTA is used since it chelates divalent ions such as Mg 2+, most
DNases require Mg2+ as a cofactor thus EDTA chelates these ions to
inhibit DNases. Therefore, degradation of the DNA is minimised. SDS
plays a role in solubilizing and denaturing of proteins as well as lysing
the lipid membrane component of the cell.

The solution was transferred into a dounce homogeniser. This is a high


pressure system that is used to break up cells. From the homogeniser,
the solution was transferred into a 250ml conical flask. To the flask, an
equal volume (40ml) of chloroform-isoamyl alcohol is added, and
stoppered with foil. Chloroform-isoamyl alcohol is an organic solvent
used to precipitate proteins, lipids and nucleases, leaving the DNA
unharmed. The flask was put into a mechanical shaker to shake for 30

The solution was transferred into a centrifuge tube and centrifuged at


2500 r.p.m. for 15 minutes. Centrifugation is used to separate fluids,
based on their density. The supernatant aqueous phase was
recovered into a 250ml conical flask. The supernatant was reextracted as above with chloroform-isoamyl alcohol, again shaking for
20 minutes and centrifuging for 15 minutes.

The supernatant was recovered into 200ml beaker. To the supernatant,


with stirring, an equal volume of cold ethanol was added. The ethanol
dehydrates the DNA so that it is easily removed from the solution and
coils around glass rod while stirring. The cold temperature prevents
the DNA from breaking apart. The DNA is collected on the glass rod.