Only ssDNA can transfer.Ī depurination step is optional. Denature the DNA (usually while it is still on the gel).įor example, soak it in about 0.5M NaOH, which would separate double-stranded DNA into single-stranded DNA. Digest the DNA with an appropriate restriction enzyme.ģ. Let's look at this technique in greater detail.ġ. This diagram shows the basic steps involved in a Southern blot. Under optimal conditions, you can expect to detect 0.1 pg of the DNA for which you are probing. The amount of DNA needed for this technique is dependent on the size and specific activity of the probe. For example, Southern Blotting could be used to locate a particular gene within an entire genome. Southern blotting is designed to locate a particular sequence of DNA within a complex mixture. To oversimplify, DNA molecules are transferred from an agarose gel onto a membrane. Southern who developed this procedure at Edinburgh University in the 1970s. Dot and slot blotting are also described.Southern blotting was named after Edward M. The third alternate protocol describes an electroblotting procedure that is currently the most reliable method for transfer of DNA from a polyacrylamide gel. Although the ease and reliability of capillary transfer methods makes this far and away the most popular system for Southern blotting with agarose gels, it unfortunately does not work with polyacrylamide gels, whose smaller pore size impedes the transverse movement of the DNA molecules. The downward capillary method described in the second alternate protocol is therefore more rapid than the basic protocol and can result in more complete transfer. This slows down the blotting process and may reduce the amount of DNA that can be transferred. The traditional method of upward capillary transfer of DNA from gel to membrane described in the first basic and alternate protocols has certain disadvantages, notably the fact that the gel can become crushed by the weighted filter papers and paper towels that are laid on top of it. A second alternate protocol describes a transfer method based on a different transfer-stack setup. The method can also be used with neutral nylon membranes but less DNA will be retained. The advantage of this combination is that no post-transfer immobilization step is required, as the positively charged membrane binds DNA irreversibly under alkaline transfer conditions. An alternate protocol details transfer using nylon membranes and an alkaline buffer, and is primarily used with positively charged nylon membranes. A Support Protocol describes how to calibrate a UV transilluminator for optimal UV irradiation of a nylon membrane. Immobilization is achieved by UV irradiation (for nylon) or baking (for nitrocellulose). With the high-salt buffer, the DNA becomes bound to the membrane during transfer but not permanently immobilized. This appendix describes Southern blotting via upward capillary transfer of DNA from an agarose gel onto a nylon or nitrocellulose membrane, using a high-salt transfer buffer to promote binding of DNA to the membrane. After immobilization, the DNA can be subjected to hybridization analysis, enabling bands with sequence similarity to a labeled probe to be identified. Southern blotting is the transfer of DNA fragments from an electrophoresis gel to a membrane support (the properties and advantages of the different types of membrane, transfer buffer, and transfer method are discussed in detail), resulting in immobilization of the DNA fragments, so the membrane carries a semipermanent reproduction of the banding pattern of the gel.
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