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. If alkaline transfer methods are used, the DNA gel is placed into an alkaline solution (typically containing sodium hydroxide) to denature the double-stranded DNA.If some of the DNA fragments are larger than 15 kb, then prior to blotting, the gel may be treated with an acid, such as dilute HCl, which depurinates the DNA fragments, breaking the DNA into smaller pieces, thus allowing more efficient transfer from the gel to membrane.The DNA fragments are then electrophoresed on an agarose gel to separate them by size.Restriction endonucleases are used to cut high-molecular-weight DNA strands into smaller fragments.
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