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
- Extraction buffer: TE buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0).
Protocol
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:
- Pick up a small amount of leaf (ca. 2 × 2 mm2) under a dissecting microscope [Note 1].
- 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.
- Add 200–400 μl of extraction buffer (TE not TE20) and mix gently [Note 2].
- Centrifuge the tubes at 10,000 × g for 30 sec at 4°C.
- Transfer the aqueous upper phase to a new 1.5 ml microcentrifuge tube.
Notes
- Samples larger than 2 × 2 mm2 are not suitable for this procedure.
- A total amount of the solution should add up to 200–450 μl.
References
- 坪田博美・有川智己. 2011. 新・分子系統解析法. 日本蘚苔類学会創立40周年記念出版委員会（編）, 改訂新版・コケ類研究の手引き, pp. 69–82. 日本蘚苔類学会, 東広島. [Tsubota, H. & Arikawa, T. 2011. Molecular phylogenetic inference. In The 40th Anniversary Commemorative Publication Committee (ed.), Methods in Bryological Research, newly revised edition. The Bryological Society of Japan, Higashi-hiroshima. (in Japanese)]
- 坪田博美・久保晴盛・大野彰洋・井上侑哉・中原-坪田美保・武内一恵・松井健一・内田慎治・向井誠二. 2013. 広島の帰化植物4．イヌカキネガラシおよびその近縁種. Hikobia 16: 321-334.
- 坪田博美・久保晴盛・向井誠二. 2009. 広島県宮島近海で見つかったヤマトウミヒルモHalophila nipponica J.Kuoについて. Hikobia 15: 339–347. [Tsubota, H., Kubo, H. & Mukai, S. 2009. A new finding of seagrass Halophila nipponica J.Kuo (Hydrocharitaceae) from Miyajima Island in Hiroshima Prefecture, SW Japan. Hikobia 15: 339–347 (in Japanese with English abstract).]
- Suzuki, T., Inoue, Y., Tsubota, H. & Iwatsuki, Z. 2013. Notes on Aptychella (Sematophyllaceae, Bryopsida): Yakushimabryum longissimum, syn. nov. Hattoria 4: 107–118.
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