The final step involves DNA precipitation to obtain pure DNA at a high concentration. Cellular components are then removed using one of the above listed technologies, for example organic extraction or silica-based technologies. The collected cells are lysed, often done chemically, using reagents such as lysozyme, EDTA, lysozyme and EDTA and other detergents, etc. An example of a commercially available kit that relies on this chemistry is the Easy-DNA® Kit from Thermo Fisher.īacterial cells are cultured in liquid media until they reach a maximum density of 2-3x10 9 cells/ml, and then harvested. This method uses hazardous organic solvents, is relatively time-consuming, and residual phenol or chloroform may affect downstream applications such as PCR. In the presence of monovalent cations such as Na +, and at -20☌, absolute ethanol efficiently precipitates polymeric nucleic acids and leaves behind short-chain and monomeric nucleic acid components, including the ribonucleotides from RNase treatment in solution. At some point in the process, RNAs are degraded through incubation with RNase. Purified DNA is usually recovered by precipitation using ethanol or isopropanol. The protein precipitate is removed following separation by centrifugation. Then, proteins are denatured/digested using a protease, and precipitated with organic solvents such as phenol, or 1:1 mixture of phenol and chloroform. In this conventional, widely used method, cells are lysed and cell debris is usually removed by centrifugation. GS FLX Titanium Rapid Library Preparation (Roche) NEBNext DNA Sample Prep Reagent Set 1 (New England Biolabs) GFX PCR DNA and Gel Band Purification (GE healthcare) Zymoclean TM Gel DNA Recovery (Zymo Research) GeneJet PCR Purification (Thermo Scientific) PureLink® PCR Purification (Thermo Fisher) Wizard ® PCR Preps DNA Purification System (Promega) Purelink Genomic DNA extraction (Thermo Fisher) Nucleon PhytoPure Genomic DNA Extraction (GE Healthcare)ĭNA Isolation for cells and tissues (Roche) NucleoSpin 8 Plant and NucleoSpin 96 Plant II, Clontech Quick-DNA Fecal/Soil Microbe Miniprep (Zymo Research)ĪccuPrep Genomic DNA Extraction (Bioneer)ĭNA Isolation for mammalian blood (Roche) Simplicity: The kit operation depends on the experience of the user, and the degree of control desired over each stage of the sample processing.īacterial Genomic DNA Mini-prep (BayGene).This is dependent upon the sample as well as the downstream applications Yield: the desired or expected amount of DNA to be purified from the sample.For example, the number of cultured mammalian cells (10 5-10 7) and bacterial cells (10 6-10 11), the weight of human tissue, plant tissue or soil, the volume of blood, or even trace DNA samples from a crime scene. Sample quantity: The kit to be used depends on the size of the sample being analysed.Humic content: If the sample has humic content such as compost, sediment and manure, a kit/method that removes humic substances should be used, as they can inhibit downstream applications like PCR.Intended use: The quality and purity of the DNA provided by the kit should be suitable for the intended downstream application, which could be sequencing, fingerprinting, PCR, quantitative PCR (qPCR), Southern blotting, random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and restriction fragment length polymorphism (RFLP) applications, restriction endonuclease digestion, or the preparation of shotgun libraries.Preparation method: Sample preparations can be: fresh or previously frozen cell pellets, paraffin-embedded or formalin-fixed tissue sections, frozen tissue sections, ethanol-fixed cells, Oragene®-preserved samples, and samples from forensic sources which might contain very limited material.Sample origin: Different kits are used to extract material from specific sources, including human tissues, blood, hair, rodent tissues, leaf tissue, bacteria, yeast, fungi, insect, stool, body fluids, spores, soil, clinical samples (e.g., biopsy samples, fine needle aspirates), forensic samples (e.g., dried blood spots, buccal swabs), and fingerprints.Factors to be considered for selecting a kit include: Basic steps involved in all DNA extraction methods.Ĭhoosing the correct DNA extraction kit can save crucial time on optimization and execution of the experiment.
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