搜搜做题作业网谁能帮我翻译一下下面这段文章?
来源：学生作业帮 编辑：搜搜做题作业网作业帮 分类：英语作业 时间：2024/05/03 20:31:51
谁能帮我翻译一下下面这段文章?
The first step in subcloning is to grow up enough material. This means either growing up a sufficient number of E. coli cells carrying the plasmid or the artificial chromosome, or infecting an E. coli culture with a phage clone. The purified recombinant vector DNA is then digested with the appropriate restriction enzyme.In some older vectors, where the insertion site is a single restriction enzyme recognition site, the insert will be flanked by sites recognized by that enzyme. The choice is then obvious. However, most commonly used vectors now have multiple cloning sites, allowing you a lot more flexibility. In particular, if it is important to be able to excise the insert in one piece, then you will need to be able to use an enzyme that does not cut the insert itself. For this purpose, you would be likely to choose an enzyme that cuts DNA infrequently such as NotI, which you can predict is unlikely to have a recognition site within the insert.On the other hand, if you want to subclone a smaller piece of the insert, you might choose a medium-frequency cutter such as EcoRI. After digestion, you would probably separate half of the reaction mixture by agarose gel electrophoresis (Figure 8.7). This can then be blotted (see the description of Southern blotting below) and probed with the same probe you used to screen the library.
With the correct DNA fragment thus identified, you can run out another gel just like the other one, cut out the DNA fragment, purify it from the agarose, and ligate it to your plasmid vector.
Artificial chromosome vectors (BAC, PAC) have inserts measuring hundreds of kilobasepairs, making it impractical to digest and separate them. An alternative approach for these is shotgun subcloning. This method is based on random physical shearing of the recombinant vector. The resulting fragments are cloned into plasmids, transformed into bacteria, and plated.A replica membrane is then lifted from this plate, and probed with the same probe. In this way, subclones containing the desired sequence can be identified.
The first step in subcloning is to grow up enough material. This means either growing up a sufficient number of E. coli cells carrying the plasmid or the artificial chromosome, or infecting an E. coli culture with a phage clone. The purified recombinant vector DNA is then digested with the appropriate restriction enzyme.In some older vectors, where the insertion site is a single restriction enzyme recognition site, the insert will be flanked by sites recognized by that enzyme. The choice is then obvious. However, most commonly used vectors now have multiple cloning sites, allowing you a lot more flexibility. In particular, if it is important to be able to excise the insert in one piece, then you will need to be able to use an enzyme that does not cut the insert itself. For this purpose, you would be likely to choose an enzyme that cuts DNA infrequently such as NotI, which you can predict is unlikely to have a recognition site within the insert.On the other hand, if you want to subclone a smaller piece of the insert, you might choose a medium-frequency cutter such as EcoRI. After digestion, you would probably separate half of the reaction mixture by agarose gel electrophoresis (Figure 8.7). This can then be blotted (see the description of Southern blotting below) and probed with the same probe you used to screen the library.
With the correct DNA fragment thus identified, you can run out another gel just like the other one, cut out the DNA fragment, purify it from the agarose, and ligate it to your plasmid vector.
Artificial chromosome vectors (BAC, PAC) have inserts measuring hundreds of kilobasepairs, making it impractical to digest and separate them. An alternative approach for these is shotgun subcloning. This method is based on random physical shearing of the recombinant vector. The resulting fragments are cloned into plasmids, transformed into bacteria, and plated.A replica membrane is then lifted from this plate, and probed with the same probe. In this way, subclones containing the desired sequence can be identified.
亚克隆的第一步是要获得足够的原料.这意味着要么通过培养携带有质粒或人工合成的染色体的大肠埃希菌（E.coli）细胞,要么用质粒转染E.coli.然后用合适的限制性酶消化纯化的重组DNA载体.在过去的载体构建中,插入位点仅是一个单独的限制酶识别的位点,这样能够保证插入的准确性.选择就是显而易见的.但是如今大多数常用的载体具有多个克隆位点,就使你的选择有更多灵活性.特别是在某些重要的试验,要对插入的一个单独片段做检测分析时,那么你就应该使用一种不会对插入的片段进行剪切的酶.为此,你可能会选择那种只能部分切割DNA的酶,比如NotI,它不太可能会识别插入部分内的所有位点.另一方面,如果你想亚克隆一小段插入片段,你可以选择一种中等剪切频率的酶,比如EcoRI.消化之后,分离出一半的琼脂糖反应混合物凝胶电泳（图8.7）.这样可以用斑点法（见下面Southern blotting的描述）和用曾经从图书馆资料中获得的探针来检测.
得到了确认的DNA序列,再使用另一部分的凝胶,切断DNA序列,通过琼脂糖纯化之后连接到质粒载体上.人工染色体载体（BAC,PAC）的插入子达到几百个碱基对,而使它接下来的消化和分离变的不容易操作.对此打靶亚克隆技术是一种有效的措施.该方法是以随机剪切获得的重组载体为基础.结果所有的克隆片段结合到相应的质粒上,通过转染细菌,在其内复制组装.从组装体中提取出复制体,再用同样的探针筛检.用此方法可以对克隆产物中所希望得到的序列进行鉴定.
希望能有所帮助!
得到了确认的DNA序列,再使用另一部分的凝胶,切断DNA序列,通过琼脂糖纯化之后连接到质粒载体上.人工染色体载体（BAC,PAC）的插入子达到几百个碱基对,而使它接下来的消化和分离变的不容易操作.对此打靶亚克隆技术是一种有效的措施.该方法是以随机剪切获得的重组载体为基础.结果所有的克隆片段结合到相应的质粒上,通过转染细菌,在其内复制组装.从组装体中提取出复制体,再用同样的探针筛检.用此方法可以对克隆产物中所希望得到的序列进行鉴定.
希望能有所帮助!