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 Growth, Treatment and TMV Inoculation Nicotiana benthamiana is to be grown under a 12 hour light and 12 hour dark cycle. Leaves of 4 week old Nicotiana benthamiana are to be treated with water alone, 0.1% ananixanthone alone or 0.1% cudraxanthone G alone. .After chemical treatment, Nicotiana benthamiana is to be inoculated with TMV infectious sap prepared from infected Nicotiana tabacum leaves. Infectious sap is prepared by grinding 1 gram of Nicotiana tabacum TMV infected leaves that show symptoms in 20 ml of 100 mM Sodium Phosphate Buffer, pH 7.2. Protocol RNA Isolation and TMV RNA Detection Total RNA is isolated from inoculated leaves using Trizol. . 0.001 mg Total RNA is to be separated on agarose gel by electrophoresis and then transferred to nylon membrane by capillary action. Nylon membrane is to be incubated with labeled DNA probes that can detect for TMV full-length genomic RNA and coat protein subgenomic RNA. Protocol Protein Isolation and TMV Coat Protein Detection Isolate total protein from Nicotiana benthamiana leaves using Tris-buffered saline (TBS) extraction buffer - 50 mM Tris-HCl and 100 mM NaCI adjusted to pH 7.5 supplemented with 1X plant protease inhibitor cocktail. Load sodium dodecyl sulfate - polyacrylamide gels with 0.005 mg of total protein and perform protein gel electrophoresisExplanation / Answer
Material required-
Nicotiana banthamiana plant, distilled water, 0.1% ananixanthone solution, 0.1% cudraxanthone G solution, TMV virus infected Nicotiana tabacum leaves, 100 mM sodium phosphate buffer (pH-7.2), total RNA isolation kit, trizol reagent, agarose gel unit, SDS-PAGE unit, blot detection instrument, 50 mM tris-HCl buffer, 100 mM sodium chloride solution, Tris buffered saline solution (pH-7.5), plant protease inhibitor cocktail solution.
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