By Jose M Martinez-Zapater, Julio Salinas
This entire number of present and crucial protocols includes many simply reproducible equipment built to be used with Arabidopsis - a method for imminent basic questions in plant biology. The tools diversity from the fundamentals of turning out to be those crops to stylish gene cloning thoughts and will, in lots of circumstances, even be utilized to different plant species with minor differences. Sections on genetics, transformation and gene expression research which are in particular worthy to scientists considering mutant research or generating and interpreting transgenic crops.
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Extra resources for Arabidopsis Protocols (Methods in Molecular Biology (Cloth))
Theor Appi Genet. 60, 197-214 34 Mathur and Koncz 4. Loewenberg, J. R. (1965) Callus cultures ofArubrdopszs Arabtdopsts If Seru 2,34 5 Moms, P C and Altmann, T (1994) Ttssue culture and transformation, m Arubzdopszs (Meyerowitz, E. , eds ), Cold Sprmg Harbor Laboratory, Cold Spring Harbor, New York, pp 173-222 6 Murashige, T and Skoog, F (1962) A revised medium for growth and bloassays with tobacco tissue cultures. Physzol Plant 15,473-497. , Hrouda, M , Bachman, A, and Schell, J (1994) Speclahzed vectors for gene taggmg and expression studies, m Plant Molecular Ezologv Manual.
Applymg gentle hand pressure on the brie stgmficantly increases the final chloroplast yield Kunst 46 5 Distribute the filtrate into 50-mL precooled tubes and centrifuge for 90 s (including deceleration) at 280g m the SS-34 rotor (see Note 3) 6 Carefully decant the supernatant, and holding the tubes upside-down, wipe then insides with paper tissue. Resuspend the crude chloroplast pellet m 1 mL of ice-cold RB using a large, natural bristle paint brush Pool the resuspended chloroplasts and rmse all the tubes and brushes wtth 1 mL of RB Add thts wash to the chloroplast suspension (see Note 4).
Of RNase stock solution ( 10 mg/mL) and incubate at 37°C for 1 h Add equal volume of 1: 1 phenol/chloroform, mix thoroughly, and spin m microcentrifuge for 10 mm. Carefully transfer the upper layer to a new tube and extract the DNA solution again with an equal volume of chloroform (see Notes 10-12) 11. Transfer the aqueous phase to another fresh Eppendorf tube, add 50 pL of 3M sodium acetate (NaOAc, pH 4 8) and 0 35 mL of lsopropanol MIX and Incubate at -2OOCfor at least 1 h (see Note 13) 12. Pellet DNA by spmnmg tube(s) m a mlcrocentrlfuge for 10 mm.
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