TITLE: Recovering Plasmid DNA from Bacterial Culture
AUTHORS: Addgene The Nonprofit Plasmid Repository

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><span>This protocol is for recovering plasmid DNA from Bacterial Culture. To see the full abstract and other resources, visit </span><a href="https://www.addgene.org/protocols/purify-plasmid-dna" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">https://www.addgene.org/protocols/purify-plasmid-dna/</span></a><span style = ":;">.</span></div></div>

[STEPS]
?. [Generalized DNA Purification]
Grow an overnight culture of bacteria.
*Pro-Tip*Refer to appropriate DNA prep protocol for volume of bacteria to grow (low copy plasmids require larger cultures).
?. [Generalized DNA Purification]
Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation.
*Pro-Tip*If your entire overnight culture cannot fit into a single centrifuge tube, aliquot it into several tubes/bottles.
?. [Generalized DNA Purification]
Remove the supernatant and resuspend the bacteria in buffer.
Note, this step gets all of the bacteria back into suspension, but within a smaller volume of buffer that is compatible with the next solution.
?. [Generalized DNA Purification]
Add a denaturing solution to the resuspended bacteria.
Note, this step causes the bacteria to lyse, releasing their contents, including plasmid DNA, into solution.
?. [Generalized DNA Purification]
Add a renaturing solution to the denatured bacteria.
Note, this step brings the pH back down causing the proteins and genomic DNA to precipitate, while leaving the smaller plasmids free in solution.
?. [Generalized DNA Purification]
Pellet the proteins and genomic DNA by centrifugation, and remove the plasmid-containing supernatant.
?. [Generalized DNA Purification]
Add either ethanol or isopropanol to precipitate the plasmid DNA.
?. [Generalized DNA Purification]
Either spin to pellet the DNA or apply the solution to a column that will bind the now precipitated DNA.
?. [Generalized DNA Purification]
Wash the pellet or column with 70% ethanol to remove excess salt.
?. [Generalized DNA Purification]
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.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Solution I - Resuspension Buffer25 mM Tris-HCl (pH 8)50 mM glucose10 mM EDTA
Store Solution I at  .
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Solution II - Denaturing Solution0.2 N NaOH1.0% SDS
Store Solution II at  .
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Solution III - Renaturing Solution (Potassium Acetate) 5M Potassium acetate glacial acetic acidof dH2O
120 ml
23 ml
57 ml
Store Solution III at  .
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Grow  overnight cultures from single colonies of bacteria containing your plasmid of interest.
2 ml
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Add  of the stock culture to a  microfuge tube.
1.5 ml
1.75 ml
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Centrifuge in microfuge tube at  for .
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Pour off the supernatant, being careful not to disturb the bacterial pellet.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Resuspend the pellet in  of cold Solution I.
100 µl
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Vortex the solution for  or until all bacteria are fully resuspended.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Add  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.
200 µl
*Pro-Tip*Do not vortex at this stage or the genomic DNA will become sheared and will therefore contaminate your purified plasmid DNA.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Incubate solution on ice for .
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Add  of cold Solution III to each tube.
150 µl
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Incubate tube on ice for .
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Centrifuge the tube for  at .
Notes: Pellet contains proteins, cell fragments, salt and other extra particles from solutions.Supernatant contains the plasmid DNA separated from bacterial chromosomes.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Collect the supernatant into a new tube by pipetting or carefully pouring.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
(Optional) Add  of 2 mg/ml RNase A to the supernatant in the new tube and incubate at  for .
5 µl
37 °C
Note, Ribonuclease A (RNase A) is a pancreatic ribonuclease that digests single-stranded RNA.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
(Optional) Perform phenol-chloroform extraction - see protocol below.
Note, phenol-chloroform extraction removes remaining contaminant proteins and RNase A from the DNA sample. When phenol is mixed with the aqueous solution containing DNA, proteins will move into the phenol phase and will be separated from the aqueous DNA.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Add either  of cold 100% ethanol or  room temperature isopropanol to the solution to precipitate the plasmid DNA; see detailed protocol section below.
700 µl
350 µl
*Pro-Tip* If precipiating with ethanol, it is often thought that an incubation of   to overnight at   or   will improve precipitation.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Pour out the supernatant.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
(Optional) Wash the pellet with 70% ethanol.
Note, this step removes excess salt from the pellet which can cause problems with some common reactions.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Air dry the pellet (can be done by inverting the tube at an angle over kimwipe) for -.
?. [Protocol: Kit-free Alkaline Lysis Plasmid Miniprep]
Resuspend pellet with - of TE.
25 µl
50 µl
?. [Protocol: Phenol-Chloroform Extraction of DNA Samples]
Add an equal volume of TE-saturated phenol-chloroform to the aqueous DNA sample.
*Pro-Tip*Water-saturated phenol-chloroform can be used if TE-saturated is not available.
?. [Protocol: Phenol-Chloroform Extraction of DNA Samples]
Vortex microfuge tube for -.
?. [Protocol: Phenol-Chloroform Extraction of DNA Samples]
Centrifuge the tube for  at  on the highest speed setting.
0 Room temperature
Note, you should see clearly separated layers:Top Phase - Aqueous DNA phaseMiddle phase - A white layer may appear, consisting of precipitated protein particlesBottom phase - Organic phase (protein)
?. [Protocol: Phenol-Chloroform Extraction of DNA Samples]
Pipet the aqueous DNA layer and place it in a new microfuge tube.
?. [Protocol: Phenol-Chloroform Extraction of DNA Samples]
Add equal volume of chloroform to the recovered aqueous DNA layer.
?. [Protocol: Phenol-Chloroform Extraction of DNA Samples]
Repeat steps 2-4.
Phenol-chloroform is a hazardous waste - DO NOT pour down sink.
?. [Protocol: Ethanol Precipitation]
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).
?. [Protocol: Ethanol Precipitation]
Invert the microfuge tube to mix.
?. [Protocol: Ethanol Precipitation]
(Optional) Place the tube either at  overnight OR  for  OR on dry ice for .
-20 °C
-80 °C
Note, this freezing may help the DNA to precipitate.
?. [Protocol: Ethanol Precipitation]
Centrifuge solution at high speed (at least ) for - at .
4 °C
Notes:Pellet contains the precipitated DNA.Supernatant contains residues, salts, and water.
?. [Protocol: Ethanol Precipitation]
Pour out the supernatant in the sink.
?. [Protocol: Ethanol Precipitation]
Open and invert the tubes on a paper towel to drain them out.
?. [Protocol: Ethanol Precipitation]
Wash pellet by adding  cold 70% ethanol.
500 µl
Note, this helps to remove excess salt from the DNA pellet.
?. [Protocol: Ethanol Precipitation]
Centrifuge solution at high speed (at least ) for  at .
0 Room temperature
?. [Protocol: Ethanol Precipitation]
Pour out the supernatant in the sink.
*Pro-Tip*Be careful, the pellet is harder to see and less well attached to the tube after the 70% ethanol wash. You can also pipet the supernatant out of the tube if you are worried about losing the pellet.
?. [Protocol: Ethanol Precipitation]
Dry with vacuum or by inverting over paper towel for -.
?. [Protocol: Ethanol Precipitation]
Resuspend dry DNA with TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA).
*Pro-Tip*DNA resuspension can take time, it is a good idea to let it sit for several hours to overnight at room temperature before quantifying and using.
?. [Protocol: Ethanol Precipitation]
Store DNA at .
4 °C