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Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well)   

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Abstract

This is a simple protocol for isolating genomic DNA from fresh plant tissues. DNA from this experiment can be used for all kinds of genetics studies, including genotyping and mapping. This protocol uses Edward’s extraction buffer to isolate DNA.

Materials and Reagents

  1. Ethanol (EtOH)
  2. Isopropanol
  3. NaCl
  4. EDTA
  5. SDS
  6. Tris (pH 7.5)
  7. Edward buffer (see Recipes)

Equipment

  1. Ceramic mortar and pestles
  2. Centrifuges (Eppendorf)
  3. Vortex (VWR International)

Procedure

  1. Add 400 μl Edward extraction buffer to a 1.5 μl tube.
  2. Take 2-3 small leaves or 1 big leaf, grind with pestle (2-3 weeks old).
  3. Vortex 5 sec, set at room temperature until all preps are ready.
  4. Spin at 16,000 rpm for 2 min.
  5. Transfer 300 μl of suspension to a fresh tube.
  6. Add 300 μl of isopropanol at room temperature for 2 min.
  7. Spin 5 min, wash pellet with 70% EtOH, and dry at room temperature.
  8. Resuspend in 100 μl H2O and store at -20 °C.
  9. Use 2 - 4 μl for PCR reaction to validate the presence of target DNA.

Recipes

  1. Edward buffer
    200 mM Tris (pH 7.5)
    250 mM NaCl
    25 mM EDTA
    0.5% SDS

References

  1. Lu, Y., Chanroj, S., Zulkifli, L., Johnson, M. A., Uozumi, N., Cheung, A. and Sze, H. (2011). Pollen tubes lacking a pair of K+ transporters fail to target ovules in Arabidopsis. Plant Cell 23(1): 81-93.
Copyright: © 2011 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Lu, Y. (2011). Extract Genomic DNA from Arabidopsis Leaves (Can be Used for Other Tissues as Well). Bio-protocol Bio101: e90. DOI: 10.21769/BioProtoc.90.
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Marisa Rosa
University of California, Berkeley
Should it not be ul instead of ml throughout the procedures?
8/12/2014 6:34:03 PM Reply
Yongxian Lu
Carnegie Institution for Science

Yes, you are right. It should be ul, instead of ml. Guess it's a typo by the editorial team. sorry for that.

8/12/2014 11:37:05 PM


Jianxin Zhao
University of Florida

Can this method could be used for extraction of Medicago or red clover ?

10/10/2016 11:48:23 AM