General description
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-minute heat inactivation step, the treated insert is combined with the ready-to-use vector and ligated in an optimized 15-minute 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 (Cat. No. 71229).
The pETBlue-1 vector is designed to identify recombinants by traditional blue/white screening while also allowing T7lac promoter based expression of target genes. Screening is independent of expression because the T7lac expression promoter is in an opposed orientation relative to the E. coli promoter that mediates blue/white screening. pETBlue-1 facilitates the expression of native unfused proteins and allows convenient subcloning of target genes already fused to existing detection and purification tags. The 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
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
* 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.
Other Notes
Novy, R.E., et al. 1996. inNovations 6, 7. Clark, J.M. 1988. Nucleic Acids Res. 16, 9677. Brownstein, J.M., et al. 1996. BioTechniques 20, 1004. Magnuson, V.L., et al. 1996. BioTechniques 21, 700. Hu, G. 1993. DNA and Cell Biology 12, 763.
Legal Information
NOVAGEN is a registered trademark of Merck KGaA, Darmstadt, Germany
Perfectly Blunt is a registered trademark of Merck KGaA, Darmstadt, Germany
Disclaimer
Toxicity: Multiple Toxicity Values, refer to MSDS
