By Jose M Martinez-Zapater, Julio Salinas
This complete number of present and crucial protocols includes many simply reproducible tools built to be used with Arabidopsis - a process for forthcoming primary questions in plant biology. The equipment variety from the fundamentals of turning out to be those vegetation to stylish gene cloning concepts and will, in lots of circumstances, even be utilized to different plant species with minor variations. Sections on genetics, transformation and gene expression research which are specifically precious to scientists all for mutant research or generating and interpreting transgenic crops.
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Extra info for Arabidopsis Protocols (Methods in Molecular Biology (Cloth))
45Mmanmtol solution and pellet the cells at 60g for 5 mm Wash the protoplast pellet by repeating this step twice (see Note 19) 11 If the protoplasts are bemg cultured m algmate drops, suspend the cells m 1 mL of algmate solutton. If the protoplasts are bemg cultured m liquid medium, resuspend the cells m 1 mL of PM medium 12. Take 5 pL from the protoplast suspension and count the number of cells m a hemocytometer (see Note 20) 13. For embedding of the protoplasts, use a wide-bore ptpet to take up the cells dispersed m sodium algmate solutton (at a density of 3-5 x lo* cells/ml) and create 250-500~pL drops on calcium-agar plates (see Notes 10 and 21).
With a 5-mL plpet, carefully transfer the supernatant mto a new, 40-mL Oak Ridge tube (see Note S), and add 3 33 mL lsopropanol MIX the solution and incubate the tube at -20°C for at least 30 mm. 6 Pellet nucleic acids by centrrfugatlon at 20,OOOg(m a Sorvall SS34 rotor) for 15 mm 7 Carefully remove supernatant and lightly dry DNA pellet by inverting the tube on paper towels for 30 min (see Note 9) 8. Resuspend the DNA pellet in 0 5-mL TE buffer 9 Transfer the DNA solution to a 1S-ml Eppendorf tube.
2 To grow plants for mesophyll protoplast isolation, transfer 5-8 7-d-old seedlings mto 250-mL Jars containing BM medium and culture them at 25°C using 16 h light and 8 h dark period (see Note 1) 3 Use leaves from 3-5-wk-old plants (see Note 2) 4. Cut leaves in half through the mid vem and then into 4-6 pieces by cross-sectionmg with a sharp blade (see Note 3) 5 Wash the cut leaves once with 0 45M sucrose solution Transfer the leaf explants into a 9-cm Petri dish and add 15 mL of enzyme solution Incubate the material for 12-16 h at room temperature in the dark (see Note 4).