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70189  pT7Blue Perfectly Blunt® Cloning Kit

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15 febrero 2012

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Número de producto Tamaño Cantidad Precio
70189-3  20 rxn 
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70189-4  40 rxn 
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Los precios están sujetos a cambios sin notificación.

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.




Producto alternativo
69839 T4 DNA Ligase
70181 NovaBlue Singles™ Competent Cells
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Productos alternativos para pT7Blue Perfectly Blunt® Cloning Kit
69839 T4 DNA Ligase
70181 NovaBlue Singles™ Competent Cells
X
Accesorios parapT7Blue Perfectly Blunt® Cloning Kit
Información sobre producto
Avoid freeze/thaw Si
Components
20 rxn40 rxn
•2 × 0.5 µg4 × 0.5 µgBlunt Vector
•10 µl10 µlPositive Control Insert
•100 µl2 × 100 µlEnd Conversion Mix
•100 U2 × 100 UT4 DNA Ligase
•1.5 ml1.5 mlNuclease-free Water
•22 × 50 µl44 × 50 µlNovaBlue Singles Competent Cells
•5 × 2 ml7 × 2 mlSOC Medium
•10 µl10 µlTest 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 NovaBlue GigaSingles™ Competent Cells, require one hour for outgrowth.
Almacenar y enviar información
Categoría de alamacenamiento ≤ -70°C
Ship Dry Ice Only
Multiple Toxicity Values, refer to MSDS

© Merck KGaA, Darmstadt, Alemania, 2012

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