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pT7Blue-3 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, also consider pSTBlue-1 Perfectly Blunt® Giga Cloning Kit (Cat. No. 71229).
The pT7Blue-3 vector is a multi-purpose cloning vector featuring a versatile multiple cloning region, blue/white screening, a T7 promoter and dual kanamycin/ampicillin resistance. Restriction sites producing 4-base 3′ overhangs are conveniently positioned at each end of the polylinker to facilitate the generation of unidirectional deletions using exonuclease III.
相关产品
70075 Introductory pT7Blue-3 Perfectly Blunt® Cloning Kit
70187 pT7Blue-3 Blunt Vector (linearized)
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
商店和运输信息
Storage ≤ -70°C
Ship Dry Ice Only
Multiple Toxicity Values, refer to MSDS |