The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable ...The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable not only for genomic DNA extraction of birch but also for that of other plants. The purity of genomic DNA extracted by the multi-times STE-CTAB extraction method is higher than that by one time STE-CTAB method, and it does not need the process of RNase. The factors of influencing ISSR system were explored based on the genomic DNA of birch extracted by the two methods. The optimal conditions for ISSR system were determined as follows: Mg2+ concentration is 1.5–3.0 mmol·L-1, dNTP concentration 0.10–0.25 mmol·L-1, the quantity of Taq polymerase 0.5–2.0 U, template DNA 30–100 ng, and the concentration of primer is 0.2?0.4 μmmol·L-1, and the reaction program was as: initial denaturation for 5 min at 94oC, 30 cycles of denaturation for 30 s at 94oC, annealing for 30 s at 51 oC, extension for 30 s at 72oC, and a final 7 min extension at 72 oC.展开更多
With Portulaca oleracea L.as an experimental material,its total DNA was extracted by the improved CTAB method,the ISSR-PCR primers were screened,and the ISSR-PCR reaction system and reaction conditions for P.oleracea ...With Portulaca oleracea L.as an experimental material,its total DNA was extracted by the improved CTAB method,the ISSR-PCR primers were screened,and the ISSR-PCR reaction system and reaction conditions for P.oleracea were Optimized.The results showed that there were 8 primers suitable for IS-SR-PCR of P.oleracea.The optimal reaction system had a volume of 25μl,including 2×Taq Platinum PCR Master Mix 12.5μl,primer 2μl,ddH_2O 9.5μl,and DNA template 1μl.The optimized ISSR-PCR of P.oleracea was started with pre-denaturation at 94℃for 360 s,followed by 30 cycles of denaturation at94℃for 60 s,annealing at 54℃for 60 s and extension at 72℃ for 90 s,and completed by extension at 72℃for 300 s.展开更多
基金This paper was supported by National Natural Science Foundation of China (No. 30571513) and National High Technology Research and Development Program of China (863 Program) (No. 2002AA241080).
文摘The basic method of DNA extraction (CTAB) was improved as the multi-times STE-CTAB extraction method and used to extract the DNA of birch leaved in this experiment. Results showed that the improved method is suitable not only for genomic DNA extraction of birch but also for that of other plants. The purity of genomic DNA extracted by the multi-times STE-CTAB extraction method is higher than that by one time STE-CTAB method, and it does not need the process of RNase. The factors of influencing ISSR system were explored based on the genomic DNA of birch extracted by the two methods. The optimal conditions for ISSR system were determined as follows: Mg2+ concentration is 1.5–3.0 mmol·L-1, dNTP concentration 0.10–0.25 mmol·L-1, the quantity of Taq polymerase 0.5–2.0 U, template DNA 30–100 ng, and the concentration of primer is 0.2?0.4 μmmol·L-1, and the reaction program was as: initial denaturation for 5 min at 94oC, 30 cycles of denaturation for 30 s at 94oC, annealing for 30 s at 51 oC, extension for 30 s at 72oC, and a final 7 min extension at 72 oC.
文摘With Portulaca oleracea L.as an experimental material,its total DNA was extracted by the improved CTAB method,the ISSR-PCR primers were screened,and the ISSR-PCR reaction system and reaction conditions for P.oleracea were Optimized.The results showed that there were 8 primers suitable for IS-SR-PCR of P.oleracea.The optimal reaction system had a volume of 25μl,including 2×Taq Platinum PCR Master Mix 12.5μl,primer 2μl,ddH_2O 9.5μl,and DNA template 1μl.The optimized ISSR-PCR of P.oleracea was started with pre-denaturation at 94℃for 360 s,followed by 30 cycles of denaturation at94℃for 60 s,annealing at 54℃for 60 s and extension at 72℃ for 90 s,and completed by extension at 72℃for 300 s.
文摘为从芒果幼叶中提取高质量的核总DNA,比较了5种DNA提取方法提取芒果叶片核DNA的效果,结果表明:改良CTAB法1提取的DNA A260/A280值最好,ISSR-PCR扩增效果最佳,是有效提取芒果基因组DNA的方法。为得到最佳的芒果ISSR-PCR反应体系,以(ATG)6为引物,采用单因素实验法,优化了ISSR-PCR反应体系:在总体积25μl的反应体系中,含1×反应缓冲液,0.20mmol.L-1dNTPs,0.20μmol.L-1引物,0.60 UTaqDNA聚合酶,30-50 ng DNA模板,不足体积用无菌超纯水补足。
