![]() To ensure the specificity of the binding of the probe to the sample DNA, most common hybridization methods use salmon testes (sperm) DNA for blocking of the membrane surface and target DNA, deionized formamide, and detergents such as SDS to reduce non-specific binding of the probe. In some cases, the hybridization probe may be made from RNA, rather than DNA. The probe DNA is labelled so that it can be detected, usually by incorporating radioactivity or tagging the molecule with a fluorescent or chromogenic dye. The membrane is then exposed to a hybridization probe-a single DNA fragment with a specific sequence whose presence in the target DNA is to be determined.The membrane is then baked, i.e., exposed to high temperature (60 to 100 ☌) (in the case of nitrocellulose) or exposed to ultraviolet radiation (nylon) to permanently and covalently crosslink the DNA to the membrane.Buffer transfer by capillary action from a region of high water potential to a region of low water potential (usually filter paper and paper tissues) is then used to move the DNA from the gel on to the membrane ion exchange interactions bind the DNA to the membrane due to the negative charge of the DNA and positive charge of the membrane. Pressure is applied evenly to the gel (either using suction, or by placing a stack of paper towels and a weight on top of the membrane and gel), to ensure good and even contact between gel and membrane. A sheet of nitrocellulose (or, alternatively, nylon) membrane is placed on top of (or below, depending on the direction of the transfer) the gel. ![]() The denaturation in an alkaline environment provides for improved binding of the negatively charged DNA to a positively charged membrane, separates it into single DNA strands for later hybridization to the probe (see below), and destroys any residual RNA that may still be present in the DNA.
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