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作 者:吉尚雷[1] 张雅斌[2] 卢玉婷[1] 陈崇洲[1] 李月红[1]
机构地区:[1]吉林农业大学,长春130118 [2]吉林省水产科学研究院,长春130033
出 处:《黑龙江畜牧兽医》2014年第5期39-41,共3页Heilongjiang Animal Science And veterinary Medicine
基 金:国家自然科学基金项目(30972191);吉林省科技发展计划资助项目(20100232)
摘 要:为了研究用脉冲场凝胶电泳技术制备高分子质量基因组DNA时的最佳条件,试验采用鲫鱼为材料,将肌肉组织研磨过滤获得细胞悬液,包被于低熔点琼脂糖包中,用蛋白酶K消化胶块后进行脉冲电泳。结果表明:采用1%琼脂糖凝胶,电泳温度为14℃,电压为4V/cm,起始和终止脉冲时间为1-40s,脉冲角度为120°,冷凝泵流量为70L/h,电泳时间为18h,能获得1000.0kb的基因组DNA,满足基因组文库构建的需要。To study the optimum conditions for using pulsed field gel electrophoresis(PFGE) technology to prepare high - molecular - weight ge- nomic DNA, the crucian carp was used as a raw material. The muscle tissue from crucian carp was grinded and filtered to obtain the cell sus- pension, and then coated with low melting - point agarose. PFGE was carried out after using the proteinase K digestion to digest the gel blocks. The results showed that the optimum reaction conditions were as follows:1% of agarose concentration, the electrophoresis temperature at 14℃, 4 V/em of the voltage imposed, 1 s of the initial switch time, 40 s of the final switch time, 120° of the pulse angle, 70 L/h of the flow of the condensate pumps, and the total electrophoresis time at 18 h. The results indicate that a 1 000 kb of genomic DNA can be obtained using the above the flow of optimum reaction conditions, which can meet the needs for construction of the genomic library.
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