The pT7Blue cloning kit features the pUC19 backbone (including high-copy number origin of replication and lac sequences), a T7 promoter, f1 origin of replication, and modified multiple cloning region. The multiple cloning region contains an EcoR V site used for T-cloning flanked by an Nde I site, which allows inserts to be conveniently subcloned into the Nde I site of many pET vectors.
- No restriction enzymes or special primers
- Compatible with any DNA polymerase
- Independent of 3ʹ-dA addition or sequence context of DNA ends
- Blue/white screening with seven different vectors, including pETBlue expression vectors
- Simple protocol takes less than one hour from PCR product to plating transformants
The Perfectly Blunt® Cloning Kits are designed for simplified cloning of DNA generated by PCR using any type of DNA polymerase. This approach enables the use of high-fidelity proofreading enzymes for amplification, thus decreasing the probability of generating mutations in the target sequence. In addition, under many conditions blunt cloning is more efficient than T-cloning, most likely due to the observation that the efficiency of single dA addition by Taq DNA polymerase varies significantly depending on the sequence context of the DNA ends, and even the number of PCR cycles performed.
These kits are also suitable for cloning restriction fragments, cDNA, or sheared DNA with the same protocols.
Storage: ≤—70 °C
