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Technique / Molecular Biology / DNA analysis techniques / DNA extraction purification and quantification
| Methods, protocols and troubleshooting on extraction, purification and quantification of high molecular weight genomic DNA, plasmid DNA, PCR products etc from tissue and cells. |
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Preparation of genomic DNA from bacteria. (Curr Protoc Mol Biol. 2001 Nov;Chapter 2:Unit 2.4.) Wilson K. Australian Institute of Marine Science, Townsville, Australia. Most protocols for the preparation of bacterial genomic DNA consist of lysis, followed by incubation with a nonspecific protease and a series of extractions prior to precipitation of the nucleic acids. Such procedures effectively remove contaminating proteins, but are not effective in removing exopolysaccharides which can interfere with the activity of enzymes such as restriction endonucleases and ligases. In this unit, however, the protease incubation is followed by a CTAB extraction whereby CTAB complexes both with polysaccharides and with residual protein, effectively removing both in the subsequent emulsification and extraction. This procedure is effective in producing digestible chromosomal DNA from a variety of gram-negative bacteria, all of which normally produce large amounts of polysaccharides. If large amounts of exceptionally clean DNA are required, the procedure can be scaled up and the DNA purified on a CsCl gradient, as described in the alternate protocol. Isolation and purification of large DNA restriction fragments from agarose gels. (Curr Protoc Mol Biol. 2002 Aug;Chapter 2:Unit 2.6.) Moore D, Dowhan D, Chory J, Ribaudo RK. Baylor College of Medicine, Houston, Texas, USA. This unit describes methods for recovering and purifying DNA restriction fragments from agarose gels. The first protocol describes electroelution of the fragment of interest from standard agarose gels using buffer-filled dialysis bags, followed by concentration and purification using an Elutip column. This approach can be used effectively for fragments of all sizes from 50 to 20,000 bp. Electrophoresis directly onto NA-45 paper (second protocol) provides relatively high yields for fragments <2000 bp. Fragments >1000 bp can also be separated on low gelling/melting agarose gels and purified by phenol extraction (third protocol), b-agarase digestion of the gel (first alternate protocol), or via glass beads extraction (second alternate protocol). Removing linkers from a fragment using a column rather than a gel is described, followed by a method for estimating DNA concentrations in solution. Purification and concentration of DNA from aqueous solutions. (Curr Protoc Mol Biol. 2002 Aug;Chapter 2:Unit 2.1A.) Moore D, Dowhan D. Baylor College of Medicine, Houston, Texas, USA. This unit presents basic procedures for manipulating solutions of single- or double-stranded DNA through purification and concentration steps. These techniques are useful when proteins or solute molecules need to be removed from aqueous solutions, or when DNA solutions need to be concentrated. The , using phenol extraction and ethanol (or isopropanol) precipitation, is appropriate for purification of DNA from small volumes (<0.4 ml) at concentrations lower than 1 mg/ml. Three support protocols outline methods to buffer the phenol used in extractions, concentrate DNA using butanol, and extract residual organic solvents with ether. An alternative to these methods is nucleic acid purification using glass beads and this is also presented. These protocols may also be used for purifying RNA. The final two alternate protocols are used for concentrating RNA and extracting and precipitating DNA from larger volumes and from dilute solutions, and for removing low-molecular-weight oligonucleotides and triphosphates. 
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Last update: 
16-May-2008 09:09 am
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An effective method of DNA extraction for bioleaching bacteria from acid mine drainage. Appl Microbiol Biotechnol. 2008 May 15;
Authors: Zeng L, Huang J, Zhang Y, Qiu G, Tong J, Chen D, Zhou J, Luo X
An effective and versatile method for microorganism lysis and direct extraction of DNA from bioleaching bacteria was developed using pure cultures and an acid mine drainage (AMD) sediment sample. In the described method, microorganisms are treated at three different incubation temperatures: boiling water incubation for 6-10min, followed by 60 +/- 5 degrees C for 30min, then 72 degrees C for 30min. The extracted DNA is purified using a phenol/chloroform/alcohol mixture and precipitated in absolute alcohol. The 16S ribosomal RNA (rRNA) and gyrB genes of the pure cultures were amplified using the polymerase chain reaction (PCR) and differentiated using repetitive intergenic DNA sequences amplification (Rep-PCR). For the AMD sediment sample, the 16S rRNA and gyrB genes of the amplicons were digested with Hin6I and MspI, and the restriction fragment length polymorphism analysis patterns were used as a fingerprint to discern community diversity. The results indicated that this method is a versatile, reproducible, effective, and rapid technique for routine DNA extraction from bioleaching bacteria. The low cost of this method also makes it attractive for large-scale studies.
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