Restriction digests of the clone give the following sizes (kb): KpnI--3.0; XbaI--3.0; BamHI--3.0. Preparation of the vector for cloning includes linearization with NarI, gel purification of the linearized vector, and treatment with T4 DNA polymerase in the presence of dATP. The target sequence can be amplified using sequence specific primers modified at the 5' end to contain an additional 13 nt complementary to the vector sequence. The forward primer should contain 5'-CTGGTTCCGGCGA-3' followed by 12-15 nt target specific sequence. The reverse primer should contain 5'-CTCGCTCCGGCGA-3' followed by 12-15 nt target specific sequence. Following amplification, the amplified sequence should also be gel purified and treated with T4 DNA polymerase in the presence of dTTP. Annealing of the vector and amplification product forms a duplex molecule that can be used directly for transformation. Sequences amplified using these primers are also compatible with the pGEM-7Zf(+)/LIC-F and pGEM-7Zf(+)/LIC-R vectors (ATCC 87048 and 87049). Ligation-independent cloning vector. |
