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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4 V/cm,起始和终止脉冲时间为1~40 s,脉冲角度为120°,冷凝泵流量为70 L/h,电泳时间为18 h,能获得1 000.0 kb的基因组DNA,满足基因组文库构建的需要。
In order to study the best conditions of pulsed-field gel electrophoresis for the preparation of high molecular weight genomic DNA, the crucian carp was used as the material to grind the muscle tissue to obtain a cell suspension, which was coated on a low melting point agarose gel and coated with proteinase K Digestion of plastic blocks after the pulse electrophoresis. The results showed that the electrophoresis temperature was 14 ℃, the voltage was 4 V / cm, the initial and final pulse time was 1 ~ 40 s, the pulse angle was 120 °, the flow rate of the condensing pump was 70 L / h , Electrophoresis time was 18 h, can get 1 000.0 kb of genomic DNA to meet the needs of genomic library construction.