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  • 實驗方法> 生物化學技術> 化學生物學實驗技術>Construction of cDNA Libraries in gt10 or gt11

    Construction of cDNA Libraries in gt10 or gt11

    關鍵詞: construction cdna libraries來源: 互聯網

    To obtain a cDNA clone of an mRNA, the mRNA must be copied faithfully into DNA, and the cDNA library must be large enough to represent the abundance class that contains the mRNA of interest. For example, in tobacco, Goldberg (1 ) has shown that it is possible to divide the mRNA population into three classes with most of the mRNAs (11,300) being in the lowest abundance class and making up 39% of the polysomal mRNA. To obtain a cDNA library that contains at least one clone for each mRNA of this class will require about 2 ? 105 clones (2 ). This can be achieved with a few micrograms of mRNA by using the efficient RNase H method of making double-stranded cDNA (3 ) and a bacterioghage λ vector that exploits the high efficiency with which in vitro packaged phage can be introduced into Escherichia coli . The choice of λ vector is important because if the DNA to be inserted makes the λ genome greater than 105% of the wild-type length, the packaged phage will have a low viability.

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