Zero Blunt™ TOPO™ PCR Cloning Kit, with pCR™-Blunt II-TOPO™ Vector, One Shot™ TOP10 Chemically Competent E. coli, and PureLink™ Quick Plasmid Miniprep Kit

The vector backbones for both of these vectors are very similar. The main difference is that the pCR4-TOPO vector has sequencing primer sites located as close as 33 base pairs from the PCR product insertion site. This minimizes the amount of vector DNA sequence that needs to be read before reaching the sequence of the insert, making the pCR4-TOPO vector very useful for sequencing applications.

The vector backbones for both of these vectors are very similar. The main difference is that the pCRII-TOPO vector is a dual promoter vector, containing the SP6 and T7 promoters for in vitro transcription/sequencing, whereas the pCR2.1-TOPO vector contains only the T7 promoter for in vitro transcription/sequencing. Both vectors contain the M13 Forward and Reverse primer sites for sequencing or PCR screening.

The sequence of the control template is proprietary.

For the Directional TOPO Cloning Vectors, a PCR product must be generated by a proofreading enzyme to create a blunt product. Pfx50 or Accuprime Pfx and Accuprime Pfx Supermix from Thermo Fisher Scientific are recommended for use.

When cloning a Pfx-amplified PCR product, the insert to vector ratio is an important consideration. The PCR product generally needs to be diluted since Pfx generates a high concentration of product and using too much insert DNA can hamper the TOPO reaction. A 1:1 molar ratio of vector to insert (or about 2-10ng of insert) is recommended.

No you cannot use pCR-Blunt or pCR-Blunt-TOPO vector to clone PCR products amplified with Taq DNA polymerase. The pCR-Blunt vector is prepared with blunt ends to accept blunt-ended fragments. Due to the terminal transferase activity of Taq DNA polymerase, PCR products amplified with this enzyme have 3'-A overhangs. In order to clone these products into pCR-Blunt, you would need to polish the ends to make them blunt (which usually is not an efficient process). Our TA Cloning Kits or TOPO TA Cloning kits are a better choice for cloning Taq-generated PCR products. TA Cloning kits include a linearized vector with 3'-T overhangs for efficient ligation of Taq-generated PCR products without additional manipulation.