By Julio Salinas, Jose J. Sanchez-Serrano
This number of conveniently reproducible Arabidopsis protocols has been up to date to mirror contemporary advances in plant biology, the of completion of the Arabidopsis genome series, that is crucial for learning plant functionality, and the improvement of complete structures techniques that permit international research of gene expression and protein and metabolite dynamics. The authors have incorporated approximately all suggestions built in Arabidopsis, others lately tailored from the conventional paintings in crop species, and the newest ones utilizing Arabidopsis as a version method. Highlights comprise the newest methods-transcriptomics, proteomics, and metabolomics - and their novel purposes (phosphoproteomics, DNA microarray-based genotyping, excessive throughput metabolite profiling, and single-cell RNA).
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Extra info for Arabidopsis Protocols, 2nd Edition (Methods in Molecular Biology)
5 m or equivalent) 2 Microfuge, centrifuge, ultracentrtfuge, and correspondmg rotors (Beckman JA- 10, JA-20, SW28, or equivalents) 3 Waring blender 4. Cheesecloth. 5. tm pore size 6 Dounce homogenizer. 7 Fme pamtmg brush 8. Tips, gloves, glass ptpets, and standard laboratory material. 3. Stock Solutions for Purification of Mitochondria The volumes of buffers indicated below are sufficient for 200-250 g fresh weight material. 1. Percoll (Pharmacta, Uppsala, Sweden) Autoclave and store at 4°C Precaution should be taken to avoid contamination of thts solution 2.
2 Transfer the powder with liquid N2 to a 40-mL Oak Ridge tube. 67 mL 5Mpotasslum acetate (no need to adJust pH) and shake vigorously. Incubate the tube on Ice for 20 mm 4 Spm tube(s) at 25,OOOg (in SS34 rotor) for 20 mm (see Note 7) Arabidopsis DNA Preparation 57 5. 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.
45M sucrose. 10 Calcium agar plates: 20 mA4 calcium chloride, 0 45M sucrose, and 1% agar Protoplast Culture and Regeneration 3. 1. 1. Leaf Mesophyll Protoplasts 1 Sterilize Arabzdopszs seeds m an Eppendorf centrifuge tube for 15 mm using 1 mL of seed sterthzmg solution Wash the seeds five times with sterile water (1 mL) and dry them by placing the open tubes in the sterile bench. 5 BM agar plates. 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.
Arabidopsis Protocols, 2nd Edition (Methods in Molecular Biology) by Julio Salinas, Jose J. Sanchez-Serrano