Recovering Plasmid DNA from Bacterial Culture

Grow an overnight culture of bacteria.

Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation.

Remove the supernatant and resuspend the bacteria in buffer.

Add a denaturing solution to the resuspended bacteria.

Add a renaturing solution to the denatured bacteria.

Pellet the proteins and genomic DNA by centrifugation, and remove the plasmid-containing supernatant.

Add either ethanol or isopropanol to precipitate the plasmid DNA.

Either spin to pellet the DNA or apply the solution to a column that will bind the now precipitated DNA.

Wash the pellet or column with 70% ethanol to remove excess salt.

Resuspend the DNA pellet, or elute the DNA off of the column using water or a neutral buffer such as TE. You will now have plasmid DNA that has been purified away from the bacterial proteins and genomic DNA. Depending on the method used, the DNA concentration and purity will vary. For more information on determining DNA concentration and purity click here.

Solution I - Resuspension Buffer

25 mM Tris-HCl (pH 8)

50 mM glucose

10 mM EDTA

Solution II - Denaturing Solution

0.2 N NaOH

1.0% SDS

Solution III - Renaturing Solution (Potassium Acetate)

120mL 5M Potassium acetate

23mL glacial acetic acid

57mLof dH₂O

Grow 2mL overnight cultures from single colonies of bacteria containing your plasmid of interest.

Add 1.5mL of the stock culture to a 1.75mL microfuge tube.

Centrifuge in microfuge tube at 10000x g for 0h 0m 30s.

Pour off the supernatant, being careful not to disturb the bacterial pellet.

Resuspend the pellet in 100µL of cold Solution I.

Vortex the solution for 0h 2m 0s or until all bacteria are fully resuspended.

Add 200µL of Solution II and invert the tube carefully 5 times to mix the contents. The contents will become clear and thicker as the proteins and DNA are denatured.

Incubate solution on ice for 0h 5m 0s.

Add 150µL of cold Solution III to each tube.

Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.

Incubate tube on ice for 0h 5m 0s.

Centrifuge the tube for 0h 5m 0s at 12000x g.

Collect the supernatant into a new tube by pipetting or carefully pouring.

(Optional) Add 5µL of 2 mg/ml RNase A to the supernatant in the new tube and incubate at 37°C for 0h 5m 0s.

(Optional) Perform phenol-chloroform extraction - see protocol below.

Add either 700µL of cold 100% ethanol or 350µL room temperature isopropanol to the solution to precipitate the plasmid DNA; see detailed protocol section below.

Pour out the supernatant.

(Optional) Wash the pellet with 70% ethanol.

Air dry the pellet (can be done by inverting the tube at an angle over kimwipe) for 0h 20m 0s-0h 30m 0s.

Resuspend pellet with 25µL-50µL of TE.

Add an equal volume of TE-saturated phenol-chloroform to the aqueous DNA sample.

Vortex microfuge tube for 0h 0m 30s-0h 1m 0s.

Centrifuge the tube for 0h 5m 0s at Room temperature on the highest speed setting.

Pipet the aqueous DNA layer and place it in a new microfuge tube.

Add equal volume of chloroform to the recovered aqueous DNA layer.

Repeat steps 2-4.

To your DNA solution, add 2-2.5 volumes 95% or 100% ethanol and 1/10 volume of 3 M Na-acetate (pH 4.8).

Invert the microfuge tube to mix.

(Optional) Place the tube either at -20°C overnight OR -80°C for 0h 30m 0s OR on dry ice for 0h 5m 0s.

Centrifuge solution at high speed (at least 12000undefine) for 0h 15m 0s-0h 30m 0s at 4°C.

Pour out the supernatant in the sink.

Open and invert the tubes on a paper towel to drain them out.

Wash pellet by adding 500µL cold 70% ethanol.

Centrifuge solution at high speed (at least 12000undefine) for 0h 5m 0s at Room temperature.

Pour out the supernatant in the sink.

Dry with vacuum or by inverting over paper towel for 0h 5m 0s-0h 20m 0s.

Resuspend dry DNA with TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA).

Store DNA at 4°C.