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本文在构建马来丝虫基因组文库的同时,对一些价廉的试剂和方法在分离纯化微丝蚴、微丝蚴DNA与pUC18 DNA片段的连接和连接后对大肠杆菌(E.coli)DH5a菌株转化、DNA重组的实验效果进行了比较。其结果:平皿孵育法比Ficoll-400×密度梯度离心法回收的微丝蚴约高一倍,纯化微丝蚴的效果相似;10×连接缓冲液的DNA重组率比5×连接缓冲液高;连接混合物转化E.coli DH5a之前加双蒸水(ddH_2O)稀释比未加ddH_2O稀释组的转化效果高一倍,DNA重组效果也比未加ddH_2O稀释的高,β-巯基乙醇(β-ME)转化E.cali DH5a的效果比二硫苏糖醇(DTT)高一倍。这些结果显示某些较为价廉的试剂可以达到和超过耗资试剂的效果。
In this paper, while constructing the genomic library of Malayan filaria, some cheap reagents and methods were used to isolate E.coli DH5a strain after isolation and purification of microfilariae, connection of microfilaria DNA and pUC18 DNA fragment The experimental results of transformation and DNA recombination were compared. As a result, the plate incubation method almost doubled the size of microfilariae recovered by Ficoll-400 × density gradient centrifugation, and the effect of purifying microfilariae was similar. The DNA recombination rate of 10 × ligation buffer was higher than that of 5 × ligation buffer; The ligation mixture was doubled before the addition of ddH 2 O into E. coli DH5a, which was twice as high as that of the ddH_2O dilution group. The DNA recombination efficiency was also higher than that without ddH_2O dilution. Β-mercaptoethanol (β-ME) The conversion of E. coli DH5a was twice as effective as dithiothreitol (DTT). These results show that some of the cheaper reagents can reach and surpass the costly reagents.