Treat the dsDNA fragment that you are using as a probe with a limiting amount of Dnase, which causes double-stranded nicks in DNA. Biotin/streptavidin detection is done by colorimetric methods, and bioluminescent visualization uses luminesence. If you used a radiolabeled 32P probe, then you would visualize by autoradiograph. Visualize your radioactively labeled target sequence. To hybridize, use the same buffer as for prehybridization, but add your specific probe.Ħ. Probing is often done with 32P labeled ATP, biotin/streptavidin or a bioluminescent probe.Ī prehybridization step is required before hybridization to block non-specific sites, since you don't want your single-stranded probe binding just anywhere on the membrane. Whatever you call it, this process relies on the ssDNA hybridizing (annealing) to the DNA on the membrane due to the binding of complementary strands. (You can also bake nitrocellulose at about 80C for a couple of hours, but be aware that it is very combustible.)ĥ. This cross links (via covalent bonds) the DNA to the membrane. (SSC provides the high salt level that you need to transfer DNA.)Īfter you transfer your DNA to the membrane, treat it with UV light. This works similarly to capillary action, except more SSC goes through the gel and membrane, so it is faster (about an hour). In this procedure, a vacuum sucks SSC through the membrane. You may use a vacuum blot apparatus instead of capillary action. Capillary action transfer draws the buffer up by capillary action through the gel an into the membrane, which will bind ssDNA. Transfer is usually done by capillary action, which takes several hours. Many scientists feel nylon is better since it binds more and is less fragile. Nitrocellulose typically has a binding capacity of about 100µg/cm, while nylon has a binding capacity of about 500 µg/cm. Traditionally, a nitrocellulose membrane is used, although nylon or a positively charged nylon membrane may be used. Transfer the denatured DNA to the membrane. Be aware, however, that the procedure may also be hampered by fragments that are too small.īe sure to neutralize the acid after this step, or the base after the prior step if you don't depurinate.Ĥ. Depurination with HCl (about 0.2M HCl for 15 minutes) takes the purines out, cutting the DNA into smaller fragments. Fragments greater than 15 kb are hard to transfer to the blotting membrane. 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. Southern blotting was named after Edward M.
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