Southern Blot Analysis - Revision history

Tkveton: /* Materials Needed */

2015-07-13T20:46:25Z

Materials Needed

← Older revision		Revision as of 20:46, 13 July 2015
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	-UV transilluminator		-UV transilluminator

			-paper towels

			-glass pipette

			-glass dishes

			-glass plate

	== Protocol ==		== Protocol ==

Tkveton: /* Protocol */

2015-07-13T20:07:45Z

Protocol

← Older revision		Revision as of 20:07, 13 July 2015
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	== Protocol ==		== Protocol ==
			-run [[Agarose Gel Electrophoresis\|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

	== Notes ==		== Notes ==

Tkveton: /* Materials Needed */

2015-07-13T19:13:24Z

Materials Needed

← Older revision		Revision as of 19:13, 13 July 2015
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	== Materials Needed ==		== 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

	== Protocol ==		== Protocol ==

Tkveton: /* Purpose */

2015-07-13T19:08:31Z

Purpose

← Older revision		Revision as of 19:08, 13 July 2015
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	{{Pagetop}}		{{Pagetop}}
	== Purpose ==		== Purpose ==
			This protocol is used to immobilize DNA fragments so the DNA can be used for Hybridization Analysis.

	== Materials Needed ==		== Materials Needed ==

Tkveton: New Template:Method Page

2015-07-13T19:07:26Z

New Template:Method Page

New page

{{Pagetop}}
== Purpose ==

== Materials Needed ==

== Protocol ==

== Notes ==

== Papers where this or a similar method has been used ==
<PMIDsummary />

== Related Ontology Terms ==

== References ==
{{RefHelp}}
<references/>

[[Category:Methods]]