Phenol Chloroform Extraction for Plasmid Isolation

• Initial protocol same as Plasmid DNA isolation.
• After the wash with 75% ethanol, suspend the pellet in 500ul of TE buffer.
• Add equal amount of phenol:chloroform:isoamyl-alcohol (25:24:1), and centrifuge at 10,000rpm for 10mins.
• Three layers will be observed, carefully take the upper aqueous layer without disturbing the middle white layer.
• Transfer that to another eppendroff and add equal volume of phenol:chloroform:isoamyl-alcohol (25:24:1), and centrifuge at 10,000rpm for 10mins.
• Again take the aqueous layer into another eppendroff and add equal amount of chloroform, centrifuge at 10,000rpm for 10mins.
• Remove the upper aqueous layer and add 1/30th volume of sodium acetate (pH5.2) and 0.7-0.8 volume of isopropanol.
• Centrifuge at 14,000 rmp for 30mins at 4 degree C.
• Carefully decant the supernatant, to the pellet add 100-200ul of 70% ethanol.
• Centrifuge at 14000 rpm for 10 mins, discard the supernatant and air dry the pellet.
• Add 50ul of TE buffer pH 8.