INTRODUCTION
• DNA is the basis of life in living forms.
• Understanding of processes of life at the DNA level is a developing field.
• Abnormalities of these processes at the level of gene and its function
are the basis of diseases.
• The gene therapy is the ultimate goal of all research on DNA.
• The extraction of DNA and its estimation is the first step in any
investigation on DNA.
• Hence it is important to be familiar with this basic step.
AIM
• To isolate DNA from the tissue by Phenol-Chloroform method.
• It is helpful to identify point sources of community and hospital based
out breaks, gene cloning, also used in forensic science for identification
of rapist, accident, war, victims, and paternity determination.
PRINCIPLE
• The cells are lysed using a detergent and then digested with the help of
an enzyme proteinase-K.
• The proteins are then separated from the cell by extracting repeatedly
with phenol and chloroform.
• Proteins being soluble in phenol/chloroform separate out as interphase
between phenol and aqueous layer after centrifugation while DNA and
RNA are contained in an aqueous layer.
• RNA is removed by RNAse treatment and DNA is precipitated out using
ethanol and sodium acetate.
PROCEDURE
Take a small pinch of tissue from biopsy sample or scrapping and mince in a
mortar and pestle using 0.5 ml of 1 x TE buffer and a pinch of sand.
Add 2 mL of 1x TE buffer and transfer the tissue homogenate to 15 ml falcon
tube. Rinse mortar with further 1.5 ml of TE buffer.
Add lysis buffer in ratio of 1:2 i.e. approximately 50% of the total volume of
the homogenate.
Add such a volume of proteinase K (10 mg/ml) that in the final volume of the
solution proteinase K is 100 g/ml.
Incubate at 37°C overnight.
Then add equal volume (~6 ml) of phenol and mix thoroughly in shaker for 15
minutes.
Centrifuge at 5000 rpm for 10 minutes at 4°C.
Take supernatant in a fresh tube using Pasteur pipette.
Add an equal volume of phenol and Chloroform isoamyl alcohol. (1:1).
Shake in shaker for 15-30 minutes. Centrifuge and take supernatant in a
fresh tube.
To the supernatant add an equal volume of CIA.
Shake for 15 min's and then centrifuge at 5000 rpm for 10 min's at 4°C.
To the aqueous supernatant add 1/10th volume of chilled 3M sodium
acetate (remove impurities) solution and 2.5 volume of chilled absolute
ethanol.
DNA forms a precipitate and can be seen as small coils that can be
spooled out in an eppendorf tube or pelleted by centrifugation.
Wash the DNA pellet with 70% ethanol and air dry.
Add 100-200 µl of 1 X buffer to dissolve the pellet.
Store DNA at -20°C to -70°C till use.
