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Plasmid DNA purification
Plasmid DNA miniprep column
Plasmid DNA purification techniques for extraction and purification of plasmid DNA from host bacteria.
DNA quality is very important factor for DNA analysis. Highly purified and intact DNA of high quality determines the success of downstream DNA analysis such as multiplex PCR, real-time quantitative PCR, DNA cloning, DNA southern blot hybridization, DNA transfection, DNA sequencing, SNPs analysis etc.
Mechanism of DNA purification (DNA isolation)
DNA can be isolated from cells, tissues, parrifin embedded samples, forensic samples, plants and a variety of organisms. Traditionally, two steps are involved in extraction and purification of DNA from samples. A highly disruptive lysis buffer is first added to break down the cellular membrane structure and separate the soluble protein and nucleic acids fraction from cell debris. The lysis buffer normally contains chotropic salts, detergents, alkaline denaturation reagents. Organic extraction using phenol:chloroform followed by ethanol precipitation is then used to extract the DNA from other soluble components. Purified DNA can be dissolved in DNase free water, or TE buffer. TE buffer may affect downstream applications due to EDTA binding to magnesium. This generic DNA extraction and DNA purification procedure apply to most downstream applications.
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In silica based DNA purification system which most of commercial kits are based on, DNA is first released by chaotropic lysis buffer disrupption followed by binding to the silica membrane in the presence of high concentration of chaotropic salts. Washes with ethanol will remove salts and other contaminants from the silica membrane binding DNA. The purified DNA is then eluted by low ionic solution such as water and TE buffer. Silica membrane based DNA purification systems are convenient for both single column purificatiton and 96 well plate automatic purification of DNA.
Plasmid purification system features
- DNA yield
- DNA purity
- DNA quality
- Time needed
- Environment harzard level
For plasmid DNA purification from host bacteria, it is critical to separate the plasmid DNA from the bacteria chromosomal DNA and cellular RNA. Several methods, including SDS/alkaline denaturation can be used to get cleared lysate that is free of bacteria chromosomal DNA and cellular RNA. This lysate clearing method utilizes the binding of linear large chromosome DNA to denatured cellular proteins after SDS-alkaline denaturation to separate them from the smaller circular plasmid DNA. Other methods to get cleared lysate are Salt/SDS precipitation, rapid boiling etc. The cleared lysates can be further purified using DNA purification techniques including silica membrane DNA purification system, organic extraction technique, differential precipitation, ion exchange chromatography etc.
General procedure of plasmid DNA purification protocol
Alkaline lysis denaturing method allows theclosed supercoiled plasmid DNA to be quickly renatured upon neutrolization, while leaving the long bacterial genomic DNA remain trapped with proteins and lipids. In the following steps the genomic DNA and proteins will be salted out in the cold.
1. Spin EP tube containing bacterial culture in LB broth for 1 min. Discard supernatant and drain tube briefly with paper towel.
2. add 0.2ml cold buffer 1 and resuspend pellet well.Note: buffer 1 contains glucose which will buffer the effect of sodium hydroxide of buffer 2.
3. add 0.4ml of buffer 2 and invert gently for few times. Sit tube at room temperature for 5 min. Note: buffer 2 contains NaOH and SDS for alkaline denaturation and break down cell membranes.
4. add 0.3ml ice-cold buffer 3 and invert gently for few times. Incubate EP tube on ice for 10 min.Note: buffer 3 contains acetic acid and potassium acetate to neutralize the alkaline solution from step 3. In this step bacterial genomic DNA will remain denaturing and bind to proteins, while smaller circled plasmid DNA will renatured and remain soluble.
5. add 1ml of ice-cold 70% ethanol and mix gently by inverting few times. Spin for 1 min to pellet DNA. Remove supernatant and drain tube with paper towel.
6. invert tube on paper towel for 5 min to drain remaining ethanol. Dissolve DNA in 50 ul TE buffer or nuclease free water. Purified plasmid DNA can be stored in definitely in -20 or -70c freezer.
Buffer:
Solution 1:per 500 ml:
| Solution 1: | per 500 ml: |
| 50 mM glucose | 9 ml 50% glucose |
| 25 mM Tris-HCl pH 8.0 | 12.5 ml 1 M Tris-HCl pH 8.0 |
| 10 mM EDTA pH 8.0 | 10 ml 0.5 M EDTA pH 8.0 |
Add H2O to 500 ml.
| Solution 2: | per 500 ml: |
| 1% SDS | 50 ml 10% SDS |
| 0.2 N NaOH | 100 ml 1 N NaOH |
Add H2O to 500 ml.
| Solution 3: | per 500 ml: |
| 3 M K+ | 300 ml 5 M Potassium Acetate |
| 5 M Acetate | 57.5 ml glacial acetic acid |
Add H2O to 500 ml.
| TE | per 100 ml: |
| 10 mM Tris-HCl pH 8.0 | 1 ml 1 M Tris-HCl pH 8.0 |
| 1 mM EDTA | 0.5 ml 0.5 M EDTA pH 8.0 |
Add H2O to 100 ml.
Resources on plasmid DNA purification
More readings on plasmid purification
Plasmid purification using non-porous anion-exchange silica fibres.
J Chromatogr A. 2007 May 18;1149(2):158-68. Epub 2007 Mar 16.PMID: 17433342
A method for plasmid purification directly from yeast.
Anal Biochem. 2002 Aug 1;307(1):13-7.PMID: 12137773
Plasmid purification using membrane-based anion-exchange chromatography.
Anal Biochem. 2001 Sep 1;296(1):138-41. No abstract available.PMID: 11520042
High-throughput plasmid purification for capillary sequencing.
Genome Res. 2001 Jul;11(7):1269-74.PMID: 11435410 [PubMed - indexed for
Immobilization of oligonucleotides on a large pore support for plasmid purification by triplex affinity interaction.
Bioseparation. 1999;7(6):317-26.PMID: 10643640
Plasmid purification using hot Mg2+ treatment and no RNase.
Biotechniques. 1999 Feb;26(2):194-6, 198. No abstract available.PMID: 10023523
A cost-effective plasmid purification protocol suitable for fluorescent automated DNA sequencing.
Mol Biotechnol. 1998 Feb;9(1):79-83.PMID: 9592771
Identification and eradication of a denatured DNA isolated during alkaline lysis-based plasmid purification procedures.
Anal Biochem. 1996 Oct 15;241(2):186-9.PMID: 8921185
A rapid plasmid purification method for dideoxy sequencing.
Methods Mol Biol. 1996;58:367-71. No abstract available.PMID: 8713886
A plasmid purification scheme for characterization of small fragments cloned into vectors.
Anal Biochem. 1995 Oct 10;231(1):267-9. No abstract available.PMID: 8678313
Plasmid purification by phenol extraction from guanidinium thiocyanate solution: development of an automated protocol.
Anal Biochem. 1991 May 1;194(2):309-15.PMID: 1713749
Generation of clonal DNA templates for in vitro transcription without plasmid purification.
Biotechniques. 1990 Mar;8(3):252-7.PMID: 2184850
Plasmid purification using reverse-phase high performance liquid chromatography resin PRP-infinity.
Nucleic Acids Res. 1988 Aug 25;16(16):8185.
Plasmid purification using high-performance gel filtration chromatography.
Nucleic Acids Res. 1988 Jun 10;16(11):5202. No abstract available.PMID: 3387230
Mechanism of autonomous control of the Escherichia coli F plasmid: purification and characterization of the repE gene product.
Nucleic Acids Res. 1988 Jan 25;16(2):413-24.PMID: 3277161
