Nucleic Acids and Oligonucleotides

Closed circular plasmid DNA (50 Mg/ml)

Choose a plasmid vector containing a single site for a restriction enzyme that generates blunt ends (e.g., SmaI, SrfI, and EcoRV). The plasmid vector and its bacterial host should carry a blue/white screening system.

Target DNA (25 Mg/ml), amplified by PCR.

When the PCR mixture contains more than one or two bands of amplified DNA, purify the target fragment by electrophoresis through low melting/gelling temperature agarose (please see Chapter 5, Protocol 6). If not purified by gel electrophoresis, PCR-amplified DNA should be prepared for ligation by extraction with phenol:chloroform and ultrafiltration through a Centricon-100 filter (please see Chapter 8, Protocol 3 ).

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