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

[DESCRIPTION]
This protocol is for recovering plasmid DNA from Bacterial Culture. To see the full abstract and other resources, visit https://www.addgene.org/protocols/purify-plasmid-dna/.

[GUIDELINES]
Tips and FAQ

Plasmid purification kits provide the fastest way to obtain a high concentration of clean plasmid DNA. To improve the purity of plasmid DNA purified without a kit it is advisable to perform a phenol/chloroform extraction of the supernatant after step 6 and before step 7. This will help to remove proteins and other contaminants from the plasmid DNA.

It is also advisable to add RNAse to the supernatant after step 6 to eliminate RNA contamination. This is included in the resuspension buffer of most kits.


[STEPS]
SECTION: Generalized DNA Purification
1. Grow an overnight culture of bacteria.
 

SECTION: Generalized DNA Purification
2. Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation.
 

SECTION: Generalized DNA Purification
3. Remove the supernatant and resuspend the bacteria in buffer.
 

SECTION: Generalized DNA Purification
4. Add a denaturing solution to the resuspended bacteria.
 

SECTION: Generalized DNA Purification
5. Add a renaturing solution to the denatured bacteria.

 



SECTION: Generalized DNA Purification
6. Pellet the proteins and genomic DNA by centrifugation, and remove the plasmid-containing supernatant.
SECTION: Generalized DNA Purification
7. Add either ethanol or isopropanol to precipitate the plasmid DNA.
SECTION: Generalized DNA Purification
8. Either spin to pellet the DNA or apply the solution to a column that will bind the now precipitated DNA.
SECTION: Generalized DNA Purification
9. Wash the pellet or column with 70% ethanol to remove excess salt.
SECTION: Generalized DNA Purification
10. 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.
SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
11. Solution I - Resuspension Buffer
25 mM Tris-HCl (pH 8)
50 mM glucose
10 mM EDTA
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
12. Solution II - Denaturing Solution
0.2 N NaOH
1.0% SDS
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
13. Solution III - Renaturing Solution (Potassium Acetate)
120 mL 5M Potassium acetate
23 mL glacial acetic acid
57 mLof dH2O
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
14. Grow 2 mL overnight cultures from single colonies of bacteria containing your plasmid of interest.

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
15. Add 1.5 mL of the stock culture to a 1.75 mL microfuge tube.


SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
16. Centrifuge in microfuge tube at   for 30 s.
SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
17. Pour off the supernatant, being careful not to disturb the bacterial pellet.
SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
18. Resuspend the pellet in 100 µL of cold Solution I.

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
19. Vortex the solution for 2 min or until all bacteria are fully resuspended.

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
20. Add 200 µL 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.
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
21. Incubate solution on ice for 5 min.

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
22. Add 150 µL of cold Solution III to each tube.

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
23. Mix by inverting several times. A white precipitate will be formed which contains the bacterial proteins and genomic DNA.
SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
24. Incubate tube on ice for 5 min.

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
25. Centrifuge the tube for 5 min at  .
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
26. Collect the supernatant into a new tube by pipetting or carefully pouring.
SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
27. (Optional) Add 5 µL of 2 mg/ml RNase A to the supernatant in the new tube and incubate at 37 °C for 5 min.
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
28. (Optional) Perform phenol-chloroform extraction - see protocol below.
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
29. Add either 700 µL of cold 100% ethanol or 350 µL room temperature isopropanol to the solution to precipitate the plasmid DNA; see detailed protocol section below.
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
30. Pour out the supernatant.
SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
31. (Optional) Wash the pellet with 70% ethanol.
 

SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
32. Air dry the pellet (can be done by inverting the tube at an angle over kimwipe) for 20 min-30 min.


SECTION: Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
33. Resuspend pellet with 25 µL-50 µL of TE.


SECTION: Protocol: Phenol-Chloroform Extraction of DNA Samples
34. Add an equal volume of TE-saturated phenol-chloroform to the aqueous DNA sample.
 

SECTION: Protocol: Phenol-Chloroform Extraction of DNA Samples
35. Vortex microfuge tube for 30 s-1 min.


SECTION: Protocol: Phenol-Chloroform Extraction of DNA Samples
36. Centrifuge the tube for 5 min at Room temperature on the highest speed setting.
 

SECTION: Protocol: Phenol-Chloroform Extraction of DNA Samples
37. Pipet the aqueous DNA layer and place it in a new microfuge tube.
SECTION: Protocol: Phenol-Chloroform Extraction of DNA Samples
38. Add equal volume of chloroform to the recovered aqueous DNA layer.
SECTION: Protocol: Phenol-Chloroform Extraction of DNA Samples
39. Repeat steps 2-4.
 

SECTION: Protocol: Ethanol Precipitation
40. 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).
SECTION: Protocol: Ethanol Precipitation
41. Invert the microfuge tube to mix.
SECTION: Protocol: Ethanol Precipitation
42. (Optional) Place the tube either at -20 °C overnight OR -80 °C for 30 min OR on dry ice for 5 min.
 

SECTION: Protocol: Ethanol Precipitation
43. Centrifuge solution at high speed (at least  ) for 15 min-30 min at 4 °C.
 

SECTION: Protocol: Ethanol Precipitation
44. Pour out the supernatant in the sink.
SECTION: Protocol: Ethanol Precipitation
45. Open and invert the tubes on a paper towel to drain them out.
SECTION: Protocol: Ethanol Precipitation
46. Wash pellet by adding 500 µL cold 70% ethanol.
 

SECTION: Protocol: Ethanol Precipitation
47. Centrifuge solution at high speed (at least  ) for 5 min at Room temperature.



SECTION: Protocol: Ethanol Precipitation
48. Pour out the supernatant in the sink.
 

SECTION: Protocol: Ethanol Precipitation
49. Dry with vacuum or by inverting over paper towel for 5 min-20 min.


SECTION: Protocol: Ethanol Precipitation
50. Resuspend dry DNA with TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA).
 

SECTION: Protocol: Ethanol Precipitation
51. Store DNA at 4 °C.