Arabidopsis gDNA isolation - Revision history

Torsten Waldminghaus: add back to protocol link

Torsten Waldminghaus — Mon, 23 Feb 2009 16:35:34 GMT

add back to protocol link

←Older revision		Revision as of 16:35, 23 February 2009
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		+	{{back to protocols}}
		+
	This is a simple and fast protocol for the extraction of genomic DNA from [[Arabidopsis thaliana]] that works fine in PCR for simple amplicons. We only use this with (rosette) leaves but it is likely that it could be used for other tissues as well.		This is a simple and fast protocol for the extraction of genomic DNA from [[Arabidopsis thaliana]] that works fine in PCR for simple amplicons. We only use this with (rosette) leaves but it is likely that it could be used for other tissues as well.

Mathias Klode: Category:DNA added

Mathias Klode — Thu, 24 Apr 2008 17:05:36 GMT

Category:DNA added

←Older revision		Revision as of 17:05, 24 April 2008
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	* 70 % ethanol		* 70 % ethanol

-	[[Category:plant]] [[Category:Arabidopsis thaliana]] [[Category:protocol]]	+	[[Category:plant]] [[Category:Arabidopsis thaliana]] [[Category:protocol]] [[Category:DNA]]

Mathias Klode: /* Material */

Mathias Klode — Thu, 24 Apr 2008 17:04:16 GMT

Material

←Older revision		Revision as of 17:04, 24 April 2008
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	* Standard reaction tubes, pipets, table top centrifuge etc		* Standard reaction tubes, pipets, table top centrifuge etc
	* Extraction buffer:		* Extraction buffer:
-	** 10 mL Tris-Cl (1 M, pH 7.5) (200 mM)	+	** 10 mL Tris-Cl (1 M, pH 7.5) (=200 mM)
-	** 2.5 mL NaCl (5 M) (250 mM)	+	** 2.5 mL NaCl (5 M) (=250 mM)
-	** 2.5 mL EDTA (0.5 M) (25 mM)	+	** 2.5 mL EDTA (0.5 M) (=25 mM)
-	** 2.5 mL SDS (10 %) (0.5 %)	+	** 2.5 mL SDS (10 %) (=0.5 %)
	** ad 50 mL (32.5 mL) H<sub>2</sub>O<sub>dd</sub> (store at room temperature)		** ad 50 mL (32.5 mL) H<sub>2</sub>O<sub>dd</sub> (store at room temperature)
	* Propane-2-ol		* Propane-2-ol

Mathias Klode: New gDNA extraction protocol for A.th.

Mathias Klode — Thu, 24 Apr 2008 17:02:39 GMT

New gDNA extraction protocol for A.th.

New page

This is a simple and fast protocol for the extraction of genomic DNA from [[Arabidopsis thaliana]] that works fine in PCR for simple amplicons. We only use this with (rosette) leaves but it is likely that it could be used for other tissues as well.

== Procedure ==
* Close the lid of a tube onto a leaf or part of a leaf.
* Add 400 µL extraction buffer (see [[Arabidopsis gDNA isolation#Material|below]]).
* Grind with micropestle until well smashed.
* Centrifuge 5 min at room temperature and maximum rpm in a standard table top centrifuge.
* Transfer 300 µL of the supernatant to a new tube.
* Add 300 µL propane-2-ol and centrifuge as before. Discard supernatant.
* Wash pellet with 70% ethanol by adding 1 mL and centrifuging again (5 min should be enough). Remove ethanol and let dry at room temperature (or, if you '''really''' are in a hurry, dry in a speedvac).
* Dissolve in 100 µL water or Tris buffer
* Use 0.5-1.0 for PCR.

== Material ==
* Standard reaction tubes, pipets, table top centrifuge etc
* Extraction buffer:
** 10 mL Tris-Cl (1 M, pH 7.5) (200 mM)
** 2.5 mL NaCl (5 M) (250 mM)
** 2.5 mL EDTA (0.5 M) (25 mM)
** 2.5 mL SDS (10 %) (0.5 %)
** ad 50 mL (32.5 mL) H<sub>2</sub>O<sub>dd</sub> (store at room temperature)
* Propane-2-ol
* 70 % ethanol

[[Category:plant]] [[Category:Arabidopsis thaliana]] [[Category:protocol]]