Description
General description
The AccepTor Vector Cloning Kits are designed to simplify the PCR cloning process by using non-proofreading thermostable DNA polymerases (i.e. KOD XL polymerase and native and recombinant Taq polymerases), which leave single 3′-dA overhangs on the reaction products. The AccepTor Vectors enable direct ligation by virtue of single 3′-dU overhangs that anneal efficiently with 3′-dA extensions on PCR products. The dU residues are converted to dT residues in vivo following transformation.
AccepTor Vectors are supplied in a ready-to-ligate format. Simply mix the vector with your PCR product, add Clonables 2X Ligation Premix (Cat No. 70573), and transform into NovaBlue Singles Competent Cells (Cat. No. 70181). The entire procedure, from PCR product to plating transformants, can be performed in as little as 40 minutes with minimal pipetting.
AccepTor Vector Kits are available in an introductory 10-reaction size as well as 20- and 40-reaction configurations. The AccepTor Vectors are also available separately without the ligation and transformation components. Also see pSTBlue-1 AccepTor Vector Giga Kit (Cat. No. 71228) for information on using higher-efficiency competent cells.
Two different vectors, pSTBlue-1 and pETBlue-1, are available as AccepTor Vector kits. Each is carefully prepared and tested for optimal cloning efficiency and provides easy visualization of recombinants by blue/white screening using lacZ α-complementation. The pSTBlue-1 is a general purpose vector with dual opposed T7 and SP6 promoters, both with amp and kan resistance, and an array of flanking restriction sites. The pETBlue-1 vector is a novel plasmid specifically designed to enable high-level T7 RNA polymerase-driven expression of target genes, while providing the convenience of a blue/white screening method. Screening is independent of expression because the associated T7lac expression promoter is in an opposed orientation relative to the E. coli promoter. pETBlue-1 facilitates the expression of native unfused proteins and allows for convenient subcloning of target genes already fused to existing detection and purification tags. An EcoR V cloning site is appropriately spaced downstream of an E. coli ribosome binding site. Inserts must encode an ATG start codon at their 5′ end if expression is desired. The sequence is numbered from the first base of the T7 promoter sequence.
In addition to the pETBlue Systems, which contain uncut plasmids, pETBlue vectors are available in formats ready for insertion of PCR products. Two types of kits are available: AccepTor Vector Kits and Perfectly Blunt® Cloning Kits. The AccepTor Vector Kit enables direct insertion of DNA containing single 3′-dA overhangs, such as those amplified with non-proofreading DNA polymerases (e.g., NovaTaq DNA Polymerase), while the Perfectly Blunt Cloning Kits are designed for cloning inserts having any type of DNA end.
Features and Benefits
No restriction enzymes or special primers
Direct ligation of PCR product with vector
Compatible with polymerases that leave single 3´-dA overhangs
Blue/white screening with pSTBlue-1 or pETBlue-1 vectors
Simple protocol takes as little as 40 minutes from PCR product to plating transformants
Other Notes
20 rxn :2 × 0.5 µg : pETBlue-1 AccepTor Vector
10 µl : AccepTor Vector Positive Control Insert
2 × 55 µl : Clonables 2X Ligation Mix
1.5 ml : Nuclease-free Water
22 × 50 µl : NovaBlue Singles Competent Cells
5 × 2 ml : SOC Medium
10 µl : Test Plasmid
2 × 0.2 ml : Tuner(DE3)pLacI Competent Cells
40 rxn4 × 0.5 : µg pETBlue-1 AccepTor Vector
10 µl : AccepTor Vector Positive Control Insert
4 × 55 µl : Clonables 2X Ligation Mix
1.5 ml : Nuclease-free Water
44 × 50 µl : NovaBlue Singles Competent Cells
9 × 2 ml : SOC Medium
10 µl : Test Plasmid
4 × 0.2 ml : Tuner(DE3)pLacI Competent Cells
Legal Information
NOVAGEN is a registered trademark of Merck KGaA, Darmstadt, Germany
Perfectly Blunt is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Commercial use of this Product requires a commercial license from EMD Millipore Corporation. Commercial use shall include but not be limited to (1) use of the Product or its components in manufacturing; (2) use of the Product or its components to provide a service, information, or data to others in exchange for consideration; (3) use of the Product or its components (or any derivatives thereof) for therapeutic, diagnostic or prophylactic purposes (including as part of a device, chip, assay or other product); or (4) resale of the Product or its components, whether or not such Product or its components are resold for use in research. Nothing contained herein shall be deemed to represent or warrant that additional third party rights are not required for use of this Product.
Toxicity: Multiple Toxicity Values, refer to MSDS
