Southern Blot Analysis

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Purpose

This protocol is used to immobilize DNA fragments so the DNA can be used for Hybridization Analysis.

Materials Needed

-DNA sample

-0.25 MHCl

-Denaturation Solution (1.5 M NaCl/0.5 M NaOH)

-Neutralization Solution (1.5 M NaCl/0.5 M Tris-Cl)

-20X and 2X SSC

-Sponge (larger than gel to be blotted)

-Whatman 3 MM filter paper sheets

-Nylon membrane

-UV-transparent plastic wrap

-UV transilluminator

-paper towels

-glass pipette

-glass dishes

-glass plate

Protocol

-run agarose gel electrophoresis on the DNA to be used

-rinse the gel with distilled water

-place gel in glass dish containing 10 gel volumes of 0.25 M HCl and shake slowly for 30 minutes at room temperature (not needed for PCR products less than 4 kb)

-pour out the HCl and rinse the gel with distilled water

-add 10 volumes of denaturation solution and shake for 20 min

-pour out the old denaturation solution and add 10 volumes fresh denaturation solution, shake for 20 minutes

-pour out the denaturation solution and rinse the gel with distilled water

-add 10 volumes of neutralization solution and shake for 20 min

-pour out the old neutralization solution and add 10 volumes fresh neutralization solution, shake for 20 minutes

-if the pH is greater than 9, rinse again with neutralization solution

-place the sponge in a glass dish and fill with 20X SSC until the sponge is half submerged

-cut three pieces of Whatman paper to the same size as the sponge, place them on top of the sponge, and wet them with 20X SSC

-take the gel out of the neutralization solution and place it on the filter paper

-roll a glass pipet over the surface to squeeze out air bubbles

-place 4 strips of plastic wrap over the edges of the gel, leaving the middle exposed

-cut the nylon membrane to fit just over the exposed part of the gel (always handle the membrane with forceps)

-submerge the membrane in 0.5 cm of distilled water and let it sit for 5 minutes

-place the membrane on the gel surface and carefully remove any air bubbles

-soak the membrane with 20X SSC

-place 5 sheets of Whatman paper cut to the side of the membrane on top of the membrane

-place 4 cm of paper towels cut to the size of the membrane on top of the Whatman paper

-lay a glass plate and a weight on top of the stack and leave overnight

-remove the weight, plate, paper towels and Whatman paper

-remove the membrane and mark with a pencil the orientation of the wells from the gel

-rinse the membrane in 2X SSC and place it on a piece of Whatman paper to dry

-wrap the membrane in plastic wrap and place it DNA-side down on a 254 nm wavelength UV transilluminator

-irradiate the membrane for the time determined in the support protocol

-membrane can be stored between 2 pieces of Whatman paper for several months at room temperature, and for longer storage should be put in a desiccator at 4 C

FOR NITROCELLULOSE MEMBRANES

-submerge the membrane in distilled water before use as with nylon, but then replace the water with 20X SSC and let soak for 10 minutes

-DO NO IRRADIATE, bake at 80 C in a vacuum for 2 hours

References

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