DNA extraction from blister rust aeciospores and urediniospores using dry grindi DNA extraction from blister rust aeciospores and urediniospores using dry grindi 来源： 互联网 802 阅读 7) ETOH precipitation: Bring volume up to 400 μl if needed using TE-8 (10 mM TRIS, 0.1 mM EDTA, pH 8.0). Add 40 μl (0.1 vol.) 3M Sodium acetate pH 5.3 and mix by inversion. Add 880 μl cold (-20 ℃) absolute ETOH and mix by inversion. Chill on ice or - 20℃ 30 min. Centrifuge at top speed 5 min. Pour off supernatant. Rinse pellet in 1000 μl cold (-20 ℃) 70% ethanol, inverting tube several times. Centrifuge at top speed 5 min., pour off, centrifuge a few seconds, pull off remaining supernatant with a pipette tip. Dry pellet 30 min. in speed vac.8) Resuspend pellet in 100 μl TE-8; heat to 55℃ 10 min. to ensure complete disassociation of pellet.9) RNA se treatment: Add 5 μl of 10mg/ml RNA se. Incubate 15 min. at 55℃, 15 min. at 37 ℃.10) Strataclean purification, 2 times: Add 14 μl fully suspended Strataclean resin slurry, mix 2 min. by flicking, centrifuge 2 min., transfer 100 μl supernatant to fresh tube, being careful not to carry over any of the resin on the last extraction.11) Final selective ETOH precipitation: To 100 μl DNA solution add 43 μl 5 M NaCl (0.427 volumes) to bring to 1.5 M NaCl concentration. Add 286 μl chilled absolute ETOH (2 volumes), hold on ice or -20℃ 30 min. Centrifuge at top speed for 5 min., rinse in 70 % ETOH as previously described, dry in speed vac and resuspend pellet in 100 μl TE-8.12) Quantification and storage: Use 2.0 μl DNA solution + 8 μl TE-8+ 2 μl dye for gel electrophoresis for initial examination of DNA concentration and quality. Use a DNA standard that has bands in the 40-60 ng range. Freeze DNA stocks at -20℃ for later use.Optional stopping spots during extraction: During ethanol precipitation, or after pellet drying. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.References for additional DNA protocols:Doudrick, R. L., W. L. Nance, C. D. Nelson, G. A. Snow, and R. C. Hamelin. 1993. Detection of DNA polymorphisms in a single urediniospore derived culture of Cronartium quercum f.sp. fusiforme. Phytopathology 83:388-392.Hamelin, R. C., J. Beaulieu, and A. Plourde. 1995. Genetic diversity in populations of Cronartium ribicola in plantations and natural stands of Pinus strobus. Theor. Appl. Genet. 91:1214-1221.Sun, L.-J., J. E. Carlson, and B. J. van der Kamp. 1995. A rapid procedure for preparing high molecular weight DNA from rust aeciospores for RAPD analysis. Proc. 4th IUFRO Rusts of Pines Working Party Conf., Tsukuba: 143-148. 您知悉并同意，丁香通平台展示产品并非为平台自身产品，您发起询价或试用申请后，平台将会把您已提供或按要求提供的相关信息同步提供至所咨询的具体产品商家，由商家为您提供具体产品的价格咨询或产品试用注意事项及相关试用产品或完成相应购买，因此您知悉并授权平台将您的信息进行同步共享。您有权拒绝，但您知悉将无法参加询价或试用活动。