Arabidopsis Protocols, 2nd Edition (Methods in Molecular by Julio Salinas, Jose J. Sanchez-Serrano PDF

By Julio Salinas, Jose J. Sanchez-Serrano

This number of with no trouble reproducible Arabidopsis protocols has been up-to-date to mirror contemporary advances in plant biology, the finishing touch of the Arabidopsis genome series, that is crucial for learning plant functionality, and the advance of complete structures methods that let worldwide research of gene expression and protein and metabolite dynamics. The authors have incorporated approximately all ideas built in Arabidopsis, others lately tailored from the normal paintings in crop species, and the latest ones utilizing Arabidopsis as a version approach. Highlights contain 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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Carefully transfer the suspension to a clean, 50-mL tube already containmg 30 mL of cold PPB and band intact protoplasts by centnfugation at 1OOgfor 10 min. 6 To recover Intact protoplasts, asptrate the buffer down to the protoplast band and collect the protoplasts with a ptpet 7 Pool the intact protoplasts, resuspend them m 10 mL of PSB, and store on ice until use 8 Before Isolating chloroplasts, collect the protoplasts by centrrfugation for 5 mm at 1OOgand resuspend at 100 pg chlorophyll/ml in cold PLB.

15 16. Dry seeds of Arabzdopszs thalzana 10% Sodium hypochlorlte solution containing 0 1% Trlton X-l 00 Eppendorf tubes. Sterilized double-dlstllled water Culture tubes 9 cm Petri dishes Pair of forceps Scissors or scalpel blades Sterile 250-mL Erlenmeyer flasks with plugs. Rotary shaker set at 120 ‘pm. Mlcrofuge. 2% gelrlte (see Note 1). 2% gelrite MSAR III (root Inducing medium). 2% gelrite 3. Methods 1. 1% Triton X-100 as surfactant and shaking for about 15 mm (see Note 2). 2. Pellet the seeds by slow centrlfugatlon in a mlcrofuge for a few seconds and remove the supernatant.

Since most plant DNA preparations are often contaminated by polysaccharides, dark adaptmg the plants for 2-3 d before collecting tissues is usually enough to reduce their content m plants. Younger leaves or other young tissues are the preferred sources for plant DNA extraction since they normally contain low amount of polysaccharides. 3. Wear gloves to handle plant tissues to avoid possible contamination 4 After collectmg tissues m liquid N,, one can allow liquid N, to evaporate and then store the samples at -70°C for later use.

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