EXTRACTION OF GENOMIC DNA

Introduction
Many techniques are currently available that allow the isolation of DNA or RNA. Most
of the procedure takes the advantage of the fact that phenol on act as an efficient deproteinisation
agent quickly disrupting cellular integrity and denaturing proteins.
Principle
This method utilizes an initial phenol extraction coupled with two phenol: chloroform
extractions to simultaneously remove proteins and lipids from nucleic acids containing solutions.
Also the composition of the aqueous interaction buffer is optimized to increase nucleic acid
recovery.
The integrity of the DNA/RNA will be improved by maintaining harvested cells or
tissues in cold.
Procedure
1. Mince the tissue and wash with saline (0.15M NaCl)
2. Homogenize the tissue in X ml of lysis buffer (10ml/g tissue)
3. Homogenize the tissue.
4. Incubate for 5 minute on ice.
5. Add SDS to a final concentration of 1% from 10% stock.
6. Mix and leave it at room temperature for 5 minutes.
7. Add equal volume of buffered phenol and swril and shake for 10 minutes.
8. Centrifuge at 10,000 rpm for 10 minutes at 4
0
C to separate the phase.
9. Centrifuge and collect aqueous phase.
10. Extract with equal volume of chloroform:isoacyl alcohol.( 24:1)
11. Centrifuge at 5000 rpm for 5 minutes.
12. Remove aqueous phase and add 1/10 volume of 3 M sodium acetate and 2.5 volume
(100ml) of cold ethanol very slowly along the sides.
13. Do not mix DNA precipitation can be seen at the junction of alcohol and the aqueous
phase.
14. Spool the DNA
OR .
15. Centrifuge at 10,000 rpm for 5 minutes. Wash with 70% ethanol ,Centrifuge at 10,000
rpm for 5 minutes and dissolve the pellet in distilled water or TE.
16. Measure the concentration by OD at 260nm and 280 nm or by diphenylamine reaction.
17. Check the DNA by running on 0.8% agarose gel electrophoresis.
Requirements
1. Lysis buffer
Components: NaCl 150mM
TrisHCl 20mM (pH 8)
EDTA - 50mM
SDS - 0.5%
Preparation: mix 15 ml of 1 M Nacl, 2ml of 1M tris (pH- 8), 10 ml of disodium EDTA
(0.5M) and 2.5 ml of 20% SDS and makeup the volume 100ml.
To be noted:
a) Disodium EDTA does not dissolve until the pH is 8. So dissolve the EDTA by
adding NaOH pellet. At point of pH 8 it will dissolve
b) Do not autoclave SDS. SDS will dissolve automatically on standing. Store at
room temperature.
2. TE buffer : TrisHCl - 10mM (pH 8)
(Tris buffer)
EDTA - 1mM

Preparation: Mix together 1ml tris buffer (pH 8) and 200 l of EDTA 0.5 M and make
up the volume to 100ml.
3. Buffered phenol l(water saturated phenol is sufficient)
4. Chloroform : isoamyl alcohol (24:1)
5. 3M sodium acetate (pH 5.2, 3M pH adjusted with glacial acetic acid)
6. Absolute alcohol
7. 70% ethanol
Result
Genomic DNA is isolated and run on 0.8% agarose gel electrophoresis, visualized and
photographed.