The TOPO® Shotgun Subcloning Kit is specifically designed for simple, rapid shotgun subcloning of large DNA, such as Bacterial Artificial Chromosome (BAC) DNA, to generate a library of clones for sequencing. The kit offers:

• An easy, effective method for shearing and blunting large DNA

• A novel topoisomerase I-activated vector for rapid ligation

• Streamlined sequence analysis of cloned DNA fragments

The TOPO® Shotgun Subcloning Kit utilizes a nebulizer (Figure 1)-a small plastic device used to atomize liquids-and compressed air to shear large DNA into 2-3 kb fragments in only 30 seconds. Once the large DNA is sheared, the ends are made blunt with T4 DNA polymerase and Klenow so it can be readily ligated into the pCR®4Blunt-TOPO® vector.The pCR®4Blunt-TOPO® vector (Figure 2) is covalently bound to topoisomerase I for high-efficiencyTOPO® Cloning (1). The pCR®4Blunt-TOPO® vector contains a minimized multiple cloning site to make sequencing clones efficient. Sequencing primer sites are located as close as 33 base pairs from the blunt cloning site, allowing you to read more of your insert sequence and less of the vector. Additional convenient features of pCR®4Blunt-TOPO® include:

• Unique Sse8387 I site in the multiple cloning site to simplify performing nested deletions prior to sequencing

• Choice of T7, T3, M13(-20) forward, and M13 reverse sequencing primer sites

• Kanamycin and ampicillin resistance genes for choice of selection in E. coli

• ccdB gene for positive selection of recombinants (2)