03. Prepare Materials and Methods Section based on the protocols Refer to the Fi
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03. Prepare Materials and Methods Section based on the protocols Refer to the Final Manuscript Guide for formatting located in CAMS Protocol - Plant Material and Growth Sow Arabidopsis thaliana Col-0, pwr-2, pi14-2, pwr-2 píf4-2. hda9-1, 35S:PIF4-HA, det1-1 35S:PIF4-HA and co1-4 35S:PIF4-HA seeds in soil and maintain under a 12 hour light and 12 hour dark cycle. . Measure hypocotyl and/or petiole growth of Col-0, pwr-2, píf4-2, pwr-2 pi14-2 and hda9-1 plants grown at 23°C or 27°C between 4 to 6 weeks of age. Protocol - Protein Isolation, Gel Electrophoresis and Detection . Isolate total protein from Col-o, 35S:PIF4-HA, det1-1 35S:PIF4-HA and cop1-4 35S: PIF4-HA plants using Tris-buffered saline (TBS) extraction buffer - 50 mM ris-HCl and 100 mM NaCI adjusted to pH 7.5 supplemented with 1X plant protease inhibitor cocktail. Load sodium dodecyl sulfate - polyacrylamide gels with 10 ug of total protein and perform protein gel electrophoresis. Transfer protein gels to nitrocellulose and incubate with 1:2000 dilution of anti-HA. Stain parallel gels with coomassie blue.Explanation / Answer
Ans 1(a) Arabidopsis thaliana is a model plant of laboratory study as it has brief life cycle, a small and descriptive genome, its compatible to tissue culture, it has particular cell type for developing roots, study of natural diversity and has mutant’s species, and can easily be genetically transformed.
Protocol: Plant material and growth
Planting in Soil Materials
• Soil
• Water
• Seeds (col-o, pwr-2,pif4-2, had 9-1, 35s:pif4-ha, det 1-1 35s:pif4-ha, cop1-4, 35s:pif4-ha)
• Pots
• lights (approximately 35-40 watt fluorescent bulbs per shelf)
• Plant shelves
• Timer for lights
Procedure
1. Fill the pots up to the brim with the soil.
2. Pots are arranged in a tray.
3. Add about 2-3 liters of water into the tray.
4. Measure about 10-20 seeds and sprinkle over the soil.
5. After covering it with the lid refrigerate tray for 3-4 days refrigerating process is called vernalization where keeping plants in the cold environment, and this helps plants to germinate. 6. After refrigeration process they are kept on racks with lights maintain 12 hour light and 12 hour dark cycle.
7. Pot lid should be present as it helps to keep the soil moist until seeds started germinating
Ans 1(b)
Measurement of hypocotyl and petiole growth of A. Thaliana plant which was grown at 250 - 270 temperature and testing was done in between 4- 6 weeks of age of plant.
Ans 2.
Protocol for Protein isolation, Gel electrophoresis, and detection:
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