Mar 24, 2023
Recovering Plasmid DNA from Bacterial Culture
Forked from Recovering Plasmid DNA from Bacterial Culture
- 1Addgene;
- 2Universitatea Bucuresti - Facultate de Biologie, IMT
External link: https://www.addgene.org/protocols/purify-plasmid-dna/
Protocol Citation: Addgene The Nonprofit Plasmid Repository, Alexandru Salceanu 2023. Recovering Plasmid DNA from Bacterial Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkw635l5r/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 11, 2023
Last Modified: March 24, 2023
Protocol Integer ID: 76849
Abstract
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.
Materials
Equipment
- Desktop microcentrifuge
- Desktop vortexer
- Vacuum (optional)
Reagents
- Overnight culture of bacteria transformed with your plasmid
- Resuspension buffer
- Denaturing solution
- Renaturing solution
- 2 mg/mL RNase A
- TE or water-saturated phenol-chloroform
- Chloroform
- 100% ethanol or isopropanol
- 90% ethanol
- 70% ethanol
- TE buffer
- 3 M Na-acetate (pH 4.8)
Generalized DNA Purification
Generalized DNA Purification
Grow an overnight culture of bacteria.
Note
*Pro-Tip*
Refer to appropriate DNA prep protocol for volume of bacteria to grow (low copy plasmids require larger cultures).
Centrifuge the culture to pellet the bacteria before proceeding with DNA preparation.
Note
*Pro-Tip*
If your entire overnight culture cannot fit into a single centrifuge tube, aliquot it into several tubes/bottles.
Remove the supernatant and resuspend the bacteria in buffer.
Note
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.
Add a denaturing solution to the resuspended bacteria.
Note
Note, this step causes the bacteria to lyse, releasing their contents, including plasmid DNA, into solution.
Add a renaturing solution to the denatured bacteria.
Note
Note, this step brings the pH back down causing the proteins and genomic DNA to precipitate, while leaving the smaller plasmids free in solution.
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.
Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
Protocol: Kit-free Alkaline Lysis Plasmid Miniprep
Solution I - Resuspension Buffer
25 mM Tris-HCl (pH 8)
50 mM glucose
10 mM EDTA
Note
Store Solution I at 4 °C .
Solution II - Denaturing Solution
0.2 N NaOH
1.0% SDS
Note
Store Solution II at Room temperature .
Solution III - Renaturing Solution (Potassium Acetate)
120 mL 5M Potassium acetate
23 mL glacial acetic acid
57 mL of dH2O
Note
Store Solution III at 4 °C .
Grow 2 mL overnight cultures from single colonies of bacteria containing your plasmid of interest.
Add 1.5 mL of the stock culture to a 1.75 mL microfuge tube.
Centrifuge in microfuge tube at 10000 x g for 00:00:30 .
Pour off the supernatant, being careful not to disturb the bacterial pellet.
Resuspend the pellet in 100 µL of cold Solution I.
Vortex the solution for 00:02:00 or until all bacteria are fully resuspended.
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.
Note
*Pro-Tip*
Do not vortex at this stage or the genomic DNA will become sheared and will therefore contaminate your purified plasmid DNA.
Incubate solution on ice for 00:05:00 .
Add 150 µL 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 00:05:00 .
Centrifuge the tube for 00:05:00 at 12000 x g .
Note
Notes:
- Pellet contains proteins, cell fragments, salt and other extra particles from solutions.
- Supernatant contains the plasmid DNA separated from bacterial chromosomes.
Collect the supernatant into a new tube by pipetting or carefully pouring.
(Optional) Add 5 µL of 2 mg/ml RNase A to the supernatant in the new tube and incubate at 37 °C for 00:05:00 .
Note
Note, Ribonuclease A (RNase A) is a pancreatic ribonuclease that digests single-stranded RNA.
(Optional) Perform phenol-chloroform extraction - see protocol below.
Note
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.
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.
Note
*Pro-Tip*
If precipiating with ethanol, it is often thought that an incubation of 00:20:00 to overnight at -20 °C or -80 °C will improve precipitation.
Pour out the supernatant.
(Optional) Wash the pellet with 70% ethanol.
Note
Note, this step removes excess salt from the pellet which can cause problems with some common reactions.
Air dry the pellet (can be done by inverting the tube at an angle over kimwipe) for 00:20:00 -00:30:00 .
Resuspend pellet with 25 µL -50 µL of TE.
Protocol: Phenol-Chloroform Extraction of DNA Samples
Protocol: Phenol-Chloroform Extraction of DNA Samples
Add an equal volume of TE-saturated phenol-chloroform to the aqueous DNA sample.
Note
*Pro-Tip*
Water-saturated phenol-chloroform can be used if TE-saturated is not available.
Vortex microfuge tube for 00:00:30 -00:01:00 .
Centrifuge the tube for 00:05:00 at Room temperature on the highest speed setting.
Note
Note, you should see clearly separated layers:
Top Phase - Aqueous DNA phase
Middle phase - A white layer may appear, consisting of precipitated protein particles
Bottom phase - Organic phase (protein)
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.
Safety information
Phenol-chloroform is a hazardous waste - DO NOT pour down sink.
Protocol: Ethanol Precipitation
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).
Invert the microfuge tube to mix.
(Optional) Place the tube either at -20 °C overnight OR -80 °C for 00:30:00 OR on dry ice for 00:05:00 .
Note
Note, this freezing may help the DNA to precipitate.
Centrifuge solution at high speed (at least 12000 rpm ) for 00:15:00 -00:30:00 at 4 °C .
Note
Notes:
- Pellet contains the precipitated DNA.
- Supernatant contains residues, salts, and water.
Pour out the supernatant in the sink.
Open and invert the tubes on a paper towel to drain them out.
Wash pellet by adding 500 µL cold 70% ethanol.
Note
Note, this helps to remove excess salt from the DNA pellet.
Centrifuge solution at high speed (at least 12000 rpm ) for 00:05:00 at Room temperature .
Pour out the supernatant in the sink.
Note
*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.
Dry with vacuum or by inverting over paper towel for 00:05:00 -00:20:00 .
Resuspend dry DNA with TE (10 mM Tris-HCl pH 8, 0.1 mM EDTA).
Note
*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.
Store DNA at 4 °C .