By Julio Salinas, Jose J. Sanchez-Serrano
This choice of effortlessly reproducible Arabidopsis protocols has been up-to-date to mirror fresh advances in plant biology, the of entirety of the Arabidopsis genome series, that's crucial for learning plant functionality, and the advance of complete platforms ways that permit international research of gene expression and protein and metabolite dynamics. The authors have integrated approximately all concepts built in Arabidopsis, others lately tailored from the conventional paintings in crop species, and the latest ones utilizing Arabidopsis as a version procedure. Highlights comprise the latest methods-transcriptomics, proteomics, and metabolomics - and their novel functions (phosphoproteomics, DNA microarray-based genotyping, excessive throughput metabolite profiling, and single-cell RNA).
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Additional info for Arabidopsis Protocols, 2nd Edition (Methods in Molecular Biology) (Не полн.)
9. 10. 11 12 13. 14. 15. 16 250 mL Sterile Erlenmeyer flasks 0 22 pm Filters for sterilization of solutions. Clnncal centrifuge (500g) Scissors and razor blades. Pair of forceps. 25, 50-, and 100~pm Sieves. 12 mL Screw-capped glass centrifuge tubes. 10 and 25 mL Sterile pipetes. Pipet pump Pasteur pipets with cotton plugs and teats Hemocytometer Inverted microscope 55 and 90 mm Petri dishes. All glassware should be sterilized prior to use and all operations are carried out usmg a sterile bench.
The activity of enzyme preparations may differ from batch to batch and thus the optimal time of mcubatlon may vary. It 1sadvisable to determine the requirements for optimal protoplasting and store a particular enzyme batch m bulk 5 At this stage of enzyme digestion, the cell suspension should be green in color and numerous spherical protoplasts should be seen floatmg under the microscope. A dirty preparation will contain many broken protoplasts and may result m low viability of surviving protoplasts 6.
Percoll (Pharmacta, Uppsala, Sweden) Autoclave and store at 4°C Precaution should be taken to avoid contamination of thts solution 2. Stock I: 1M MOPS solution (without adJustmg pH) 3. Stock II* 5M KOH 4. Stock III: 100 mM EGTA-KOH pH 7 2. 2. 4 (with 5M KOH), autoclave, and store at 4°C. Before use, add dtthiotreitol (DTT) to a final concentration of 10 mA4, bovine serum albumin (BSA) to 2 g/L and polyvmylpyrrolidon (PVP) to 6 g/L 7. 2 (with 5M KOH), autoclave, and store at 4°C. Before use, add BSA to 1 g/L.