Rapid method for introducing restriction sites into double stranded plasmid DNA.

Cloning sites in double stranded expression vectors are often times incompatible with the fragment to be subcloned, or in a case such as ours, a blunt end site and a site for an enzyme which cuts infrequently in the target DNA are not present in the shuttle vector but are required for high efficiency blunt end ligation (1). The following is a rapid method for introducing new restriction sites into virtually any double stranded plasmid containing at least one site that produces a 5' overhang and one that produces a 3' overhang in the cloning region. A single stranded oligonucleotide containing the new restriction sites is synthesized such that one end contains complementary sequence to the 5' overhang and the other end contains complementary sequence to the 3' overhang. Producing and annealing the complementary oligonucleotide is unnecessary, and a crude preparation of oligonucleotide is sufficient, as only the full length oligonucleotide will contain both cohesive ends. 1 uL (3.3 ug) of the oligonucleotide (Synthecell, machine grade) is phosphorylated by adding 14 uL of 5X (BRL), 42 uL of ddH2O, 10 uL of 10 mM ATP, and 3 uL of T4 polynucleotide kinase (BRL). After a 30 min incubation at 370C, the enzyme is inactivated at 650C for 10 min. 1 uL (50ng) of the phosphorylated oligonucleotide, 2 uL (500ng) of EcoRI/PstI double digested vector (gel purified but not dephosphorylated), 2 uL of 5X ligase buffer (BRL), 1 uL of ddH O, and 1 uL (1U) of T4 DNA ligase are gently mixed together and incubated at 220C for up to 4 hrs. The ligation reaction is diluted 1:5 with ddH20 and luL used in a standard bacterial transformation reaction. Recombinant plasmids are identified by colony lift hybridization using the oligonucleotide as the probe. A restriction digest analysis confirms that the ligation has been successful (Fig 1). We used the following 27mer to introduce new NotI, SmaI, NcoI, and SfiI sites while preserving the original EcoRI and PstI sites in the vector: SfiI