Arabidopsis gDNA isolation: Difference between revisions
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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. | ||
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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 | ||
* 70 % ethanol | * 70 % ethanol | ||
[[Category:plant]] [[Category:Arabidopsis thaliana]] [[Category:protocol]] | [[Category:plant]] [[Category:Arabidopsis thaliana]] [[Category:protocol]] [[Category:DNA]] | ||
Latest revision as of 09:35, 23 February 2009
| 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.
Procedure
- Close the lid of a tube onto a leaf or part of a leaf.
- Add 400 µL extraction buffer (see 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) H2Odd (store at room temperature)
- Propane-2-ol
- 70 % ethanol
