Protocol by Tsubota et al 2013

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A simple protocol for DNA extraction used in the study by Tsubota et al. (2013)

Reagents and solutions

  1. Extraction buffer: TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0).


This protocol is for DNA isolation using small quantities of materials, and a modification of the simple chloroform method devised by Tsubota et al. (2009), Tsubota & Arikawa (2011) and Suzuki et al. (2013). The method consists of the following steps:

  1. Pick up a small amount of leaf (ca. 2 × 2 mm2) under a dissecting microscope [Note 1].
  2. Grind the lamina with 30 μl extraction buffer (TE or TE20 buffer; see recipe, above) using a polypropylene pestle in a 1.5 ml microcentrifuge tube.
  3. Add 200–400 μl of extraction buffer (TE not TE20) and mix gently [Note 2].
  4. Centrifuge the tubes at 10,000 × g for 30 sec at 4°C.
  5. Transfer the aqueous upper phase to a new 1.5 ml microcentrifuge tube.


  1. Samples larger than 2 × 2 mm2 are not suitable for this procedure.
  2. A total amount of the solution should add up to 200–450 μl.


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