Phenol-chloroform DNA extraction from Sporosarcina pasteurii

Grow Sporosarcina pasteurii cells in 150 mL of BHI/330 mM urea at 30°C with 200 rpm shaking to an OD600 of 3.5.

Concentrate cells by centrifugation at 15,000 x g for 10 min and pour off the supernatant.

Resuspend cells in 10 mL of Resuspension Buffer (50 mM Tris pH 8.0, 10% sucrose).

Add 2.5 mL Lysis Buffer (Tris pH 8.0, 10 mg/mL lysozyme), 1.5 mL 10% SDS, 5 µL 100 mg/mL RNase A, and 25 µL 50 mg/mL proteinase K to lyse cells and stabilize DNA.

Incubate for 1 h at 37°C.

Add 1.3 mL of 3.0 M sodium acetate (pH 5.5) and 30 mL of 100% ethanol to separate DNA from other biomacromolecules by precipitation.

Spool DNA onto a glass hook and transfer to 20 mL of TE buffer.

Dissolve DNA in TE buffer for 1 h at 37°C.

Add a 5 mL aliquot of DNA to 7 mL of phenol:chloroform:isoamyl alcohol (25:24:1, Saturated with 10mM Tris, pH 8.0, 1mM EDTA) and mix by inversion.

Separate phases by centrifugation at 10,000 x g for 10 min.

Pipette off the aqueous phase and add to 5 mL of chloroform. Mix by vortexing.

Separate phases by centrifugation at 10,000 x g for 10 min.

Pipette off the aqueous phase and add to 30 mL of 100 % ethanol. Mix by inversion.

Collect precipitated DNA by centrifugation at 10,000 x g for 10 min.

Wash pelleted DNA twice by adding 5 mL of 70% ethanol, mixing by inversion, centrifuging at 10,000 x g for 5 min, and removing the supernatant.

Allow the pellet to dry.

Dissolve the final pellet in 500 µL of TE buffer at 37°C for 1 h.