The principle and procedures of RLGS method was first described by Hatada et al. (1991) and its improvement was described by Asakawa (1996). Basically based on their procedures, we have successfully applied RLGS to gentic analysis in plants (Kawase, 1996; Kawase et al., 1999). Our current RLGS protocol is described here. A. Preparation of DNA Solution In the case of rice, for exampleThis method may be appllicable for many grass species and some other plants. More simplified emthod for wheat DNA is here. Collect 2-10 g fresh leaves, cut them into pieces (about 1 cm long) and freeze by liquid nitrogen mill frozen leaf pieces with dry ice using a foods-mill (IWATANI Milser IFM-150D) and collect the flour into a 50 ml tube store leaf flour at -40oC until extraction. Add 20 ml preheated (to about 70oC by micro-oven) extraction buffer A and 50 ul proteinase K (20 mg/ml), and then stir gently using a spatula. Incubate at 55oC for 30 min. |
In the case of egg plant, for exampleThis method may be succesfully appllicable for other plants especially when the above-mentioned method gives a dirtily colored DNA solution because of a large amount of polyphenolic substances in the plant organs.B. Preparation of gel holder |
C. Preparation of 1-D GelD. Not I and EcoR V digestionE. LabellingF. Preparation of 2-D GelG. in situ digestion |
H. Running 2-D gel electrophoresisI. Gel dryingJ. Autoradiography |
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