Extraction of DNA from Virus
Mix Lysing Solution ( LS ) & Sodium Perchlorate ( SP ) in 2 : 1 .
Mix the combined Lysing Solution & sodium Perchlorate with virus prep : 18.5 ml LS + SP with 10ml virus prep ( ~ 10¹¹ PFU / ml ) .
Incubate the lysate at 55ºC for 1hrs .
Add 30ml of phenol : [ chloroform : isoamyl alcohol ( 25 : 1 ) ] in the proportion equal to 1 : 1 .
Shake them by hands for 5mins .
Transfer the liquid to the centrifuge tubes , balance them & centrifuge for 15 mins at 14000 rpm at 4ºC .
Transfer the aqueous ( upper ) part with pipette to the clean centrifuge tubes .
Don’t worry getting protein in it .
Put the bottom layer into the waste container .
Centrifuge tubes again at 14,000 rpm for 15 min .
Now it easy to get pure liquid without protein debris .
Aqueous layer may be stored overnight at 4 ºC .
Estimate the volume of the aqueous layer and add 0.1 volume of 3M ( or 5M ) Sodium Acetate ( pH 6.0 ) .
Collect DNA by swirling glass rod .
Let it dry on the glass rod .
Pour off alcohol into the waste container .
Put DNA on the rod into the beaker & add 10 to 20ml of cold 76 % Ethanol ( - 20ºC ) .
Let it stand for 10 - 20 min .
Press the glass rod with DNA against the wall of beaker to get rid of the ethanol .
Invert the stirring rod in the test tube rack and dry for 5 min .
Use 15ml plastic tubes .
Dissolve DNA in 4ml of TE .
Estimate DNA concentration using spectrophotometer or gel to ensure DNA quantity .
Gently overlay the aqueous layer with 2 volumes of 95 % of ethanol .
