DNA can be isolated from cells and tissues by high salt or phenol extraction.
Here, chromatin and proteins are dissociated by treatment with SDS. The proteins
are denatured by phenyl chloroform treatment. It becomes insoluble and can be
prepared by the following chemicals.
- pH-8.0 (0.9% NaCl and 0.01 M EDTA)
- 10% SDS
- 80% phenol
- 95% ethanol
- 2.5 gm of liver tissue was homogenized in 25 mL of saline EDTA and
transferred to a 250-mL conical flask.
- To this, 2.5 mL of 10% SDS and 2.5 mL of 80% phenol was added and
shaken well for 10 minutes.
- The entire mixture was centrifuged at 10000 rpm for 10 minutes. The
aqueous layer was separated out.
- To the remaining material, (interphase + phenol phase), equal volume of
chloroform, isoamyl alcohol (24 : 1) was added and shaken for 25 minutes.
- It was again centrifuged at 10000 rpm for 10 minutes.
- The aqueous phase was collected.
- To the aqueous phase, equal volume of 80% phenol was added and again
centrifuged at 10000 rpm for 10 minutes.
- To the aqueous phase obtained, 95% ice cold ethanol was added and then
the flask was shaken gently.
- The DNA are precipitated as a white mass.
By the addition of ice-cold ethanol, DNA strands were obtained as a white
mass or turbidity.