Clone entry

The reaction differs from excision of the X chromosome because the Entry Clone contains two attL sites and the destination vector contains two attR sites (Hartley et al., 2000). The att sites are mutated to ensure recombination only occurs between attLl and attRl and between attL2 and att.R2. The recombination reaction proceeds through a cointegrate molecule that is resolved to create a destination vector containing the gene of interest with the desired promoter and tag sequences (Fig. 4.6). 

Figure 4.6. Recombinational cloning (RC). A. Cloning of PCR products using the attB + attP > attL + attR reaction catalyzed by Int and IHF. The result is an Entry Clone that can be used to create functional vectors. B. Conversion of an Entry Clone to a functional vector using the attL + attR > attB + attP reaction catalyzed by Int, Xis and IHF. A wide variety of functional vectors can be constructed by using a Destination Vector with the appropriate promoters and tags.

The most efficient strategy may be to use the topoisomerase cloning method in conjunction with the Crs-lox or X recombination systems. In this way, PCR products can be efficiently inserted into the Univector or Entry Clone with minimal additional sequences added to PCR primers. Once the genes are inserted into the starting vectors they can rapidly be converted to functional vectors using either Cre-lox or X recombination. 

Cloning-Ready/ Entry Clone plasmid minipreps... 

The BP reaction is a recombination reaction that transfers a DNA fragment (such as a PCR product) flanked by attB recombination sites into an E. coli vector, called Donor vector containing attV sites, to produce a new plasmid, called Entry clone. This reaction is catalyzed by the BP Clonase mix of recombination proteins. In this reaction, the ccdE gene is removed from the Donor vector, thereby allowing the transformants to grow. 

The concentration of an Entry clone plasmid should be kept at a low level (1-10 ng/pi) in the LR reaction. A high amount of an Entry clone proved to be adverse, contrary to our expectations. Similarly, the concentration of the destination vector should be kept low. In contrast to the LR reaction, it would be better to use the insert DNA (PCR products) at a high concentration in the BP reaction. 

Gateway entry clones 611 Gateway, multisite 616 Gateway reactions 610... 

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