Plasmids genomic library construction

Construct a large-insert genomic library in a broad host range plasmid. This will facilitate the cloning of CO-shift related genes as well as be useful in complementation experiments when a CO-shift mutant is obtained. 

Figure 1.2-1. Examples of vectors used for construction of genomic libraries (maximum capacity 24kb). Standard X vectors (XEMBL3, XGEMll, X.DASH, A,EMBL3, and A.SK6) and diphasmid vectors (XSK17 and XSK40) are shown. Sizes are in kilobases and are not shown in the scale. Not all restriction sites are shown. Heavy thick lines represent stuffer fragments open boxes mark plasmid and M13 sequences /acZO is the lac operator sequence. T3, T7, SP6-promoters for T3, T7, and SP6 RNA polymerases.

In some cases, it is more convenient to work with a genomic library in plasmid than in lambda phage form. The construction of representative genomic library directly in a plasmid vector has several drawbacks and difficulties. However, these problems can be easily solved with the help of phasmid and diphasmid vectors. In this case, a genomic library is constructed in lambda phage (e.g., SK40), and then the whole library is converted to plasmid form. 

An example of the former cloning strategy, outlined in Fig. 6.17, is provided by Rishi McManus (695), who have constructed a small, size-selected genomic DNA library in a plasmid vector of Escherichia coli using total DNA isolated from Echinococcus granulosus. Subsequent differential... 

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