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allan2009 allan2009
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Posts: 31
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13 years ago
A directional (forced) clonning is planned using the restriction enzymes Pst1 and Sph1 to generate incompatible termini in the vector pUC18. Note that enzymes have different optimum buffer requirements and must be digested sequentially rather than simultaneously. The plasmid is first digested with Sph1, purified on a column, eluted and resuspended in Pst1 buffer and digested with Pst1. However, when this plasmid was used in the clonning experiment with linear inserts having Sph1 and Pst1 ends, no recombinant plasmid clones were obtained? Not sure why, and how to correct.
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