If you have a single colony of gram positive bacteria, how will you purify genomic DNA from it? What is the method of DNA quantification
Answers
Answer:
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Answer:
DNA extraction is required for a variety of molecular biology applications. Figure 1 lists the basic steps involved in all DNA extraction methods. Many commercial kits are available to isolate DNA from a variety of biological materials [1, 2]. The sensitivity of polymerase chain reaction (PCR) detection has been shown to be different for various DNA kits [3]. Therefore, selecting the best methodology for your application is crucial.
Explanation:
Choosing the correct DNA extraction kit can save crucial time on optimization and execution of the experiment. Factors to be considered for selecting a kit include:
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 [1, 2].
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
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.
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.
Sample quantity: The kit to be used depends on the size of the sample being analysed. For example, the number of cultured mammalian cells (105-107) and bacterial cells (106-1011), the weight of human tissue, plant tissue or soil,, the volume of blood, or even trace DNA samples from a crime scene.
Yield: the desired or expected amount of DNA to be purified from the sample. This is dependent upon the sample as well as the downstream applications
Simplicity: The kit operation depends on the experience of the user, and the degree of control desired over each stage of the sample processing.
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