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pT7Blue Perfectly Blunt® Cloning Kit
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 (Novy 1996, Clark 1998, Brownstein 1996, Magnuson, 1996, Hu 1993).
With the Perfectly Blunt cloning protocol, you can go from PCR product to plating transformants in less than one hour with minimal hands-on time. The finished PCR product is converted to a blunt, phosphorylated form in a 15-minute reaction using premixed reagents. Following a 5-min heat inactivation step, the treated insert is combined with the ready-to-use vector and ligated in an optimized 15-min reaction. An exclusive 8-minute transformation procedure using highly efficient NovaBlue Singles™ Competent Cells (Cat. No. 70181) generates recombinant colonies that are easily visualized by blue/white screening.
Note that the Perfectly Blunt method is not limited to cloning PCR products; these kits are also suitable for cloning restriction fragments, cDNA, or sheared DNA with the same protocols.
Seven different vectors are available in Perfectly Blunt® Cloning Kits. Vector choices include those designed for general cloning, sequencing, optimal in vitro transcription/translation, and optimal protein expression in E. coli. Each vector is available in a kit containing sufficient reagents for 10, 20, or 40 reactions.
“Vector only” kits are also available in 20- and 40-reaction sizes without ligase and competent cells. For higher-efficiency competent cells, see also pSTBlue-1 Perfectly Blunt Giga Cloning Kit 71229).
The pT7Blue vector 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.
其他选项
69839 T4 DNA Ligase
70181 NovaBlue Singles™ Competent Cells
相关产品
70174 pT7Blue Blunt Vector (linearized)
70183 Introductory pT7Blue Perfectly Blunt® Cloning Kit
产品信息
Avoid freeze/thaw 是的
Components
20 rxn 40 rxn
• 2 × 0.5 µg 4 × 0.5 µg Blunt Vector
• 10 µl 10 µl Positive Control Insert
• 100 µl 2 × 100 µl End Conversion Mix
• 100 U 2 × 100 U T4 DNA Ligase
• 1.5 ml 1.5 ml Nuclease-free Water
• 22 × 50 µl 44 × 50 µl NovaBlue Singles Competent Cells
• 5 × 2 ml 7 × 2 ml SOC Medium
• 10 µl 10 µl Test Plasmid
Features and benefits
No restriction enzymes or special primers
Compatible with any DNA polymerase
Independent of 3′-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
* All times listed refer to kits using NovaBlue Singles™ Competent Cells and ampicillin or chloramphenicol selection. Giga kits, which use No |