Allow rapid and directional cloning of PCR fragments into powerful vectors for mammalian expression or in vitro expression.

• Fast, convenient and directional PCR cloning
• No need for restriction or ligation enzymes
• Cloning efficiency is greater than 85%
• Powerful pPK-CMV vectors for high-level gene expression in mammalian cells 
• Available for native or HA-tagged protein expression

The FastClone PCR cloning method saves hours of work because it eliminates the need for restriction or ligation enzymes. PCR-amplified genes are simply mixed with linearized FastClone vectors and transformed into highly competent E. coli DH5α. Typically, more than 85% of the resultant colonies contain clones with the relevant insert in the correct orientation for expression.
Because the FastClone vectors do not need multiple cloning sites or other enzyme recognition sites (e.g., a topoisomerase I recognition site), no unwanted sequences are incorporated into the resulting expression vector. This is especially important when it is necessary to minimize the risk of structural or functional interferences. The pPK-CMV FastClone vectors included in the kit contain an efficient SV40 terminator as well as a modified human cytomegalo-virus (CMV) based promoter/enhancer sequence that has been optimized for high-level expression in different mammalian cells.
Three versions of the FastClone PCR Cloning Kit are available:

The FastClone PCR Cloning Kit 1 contains the linear expression vector pPKCMV-E1/FC without any tag.

The FastClone PCR Cloning Kit 2 contains the linear expression vector pPKCMV-E2/FC with an N-terminal HA fusion tag.

The FastClone PCR Cloning Kit 3 contains the linear expression vector pPKCMV-E3/FC with a C-terminal HA fusion tag.

Each kit also contains a FastClone CAT  control template.

PromoKine OptiCells Competent E. coli Cells are recommended for use with the FastClone PCR Cloning Kits. These specially optimized competent cells provide transformation efficiencies of >1x10⁹ cfu/μg, even under the most difficult transformation conditions, such as when a large amount of DNA is used for transformation, when diluted DNA samples are used, or when a high salt ligation mix is used. OptiCells Competent E. coli Cells are available separately.

See also: Cell Transfection Reagents

• Excellent gene expression in vitro and in vivo
• Easy and adaptable for all genes
• Rapid addition of N-terminal hemagglutinin (HA) eptitope tag to genes of interest (FastExpress Kit HA) simplifies detection of expressed proteins
• High-throughput screening of functional gene expression
• Rapid screening of genes for intracellular localization studies

 The FastExpress Gene Expression System is an innovative method for high-throughput gene expression. It makes time-consuming and tedious gene cloning unnecessary by adding a strong eukaryotic promoter and terminator sequence to any target gene of interest via a simple 2-step PCR procedure.
The linear FastExpress fragments generated by the FastExpress 2-step PCR are transcriptionally active and can be expressed in vitro and in vivo. The obtained expression levels are similar to those typically observed with conventional plasmid vectors.
Because FastExpress eliminates the need for classic gene cloning, bacterial transformations, and plasmid purifications, large numbers of genes can be shotgun expressed and examined simultaneously. This allows for extremely quick experiments with very little processing time.
The FastExpress Gene Expression Kits are available in two versions. One generates a FastExpress fragment without a tag. The other can be used to add an N-terminal hemagglutinin (HA) tag to the 5’ end of FastExpress-amplified genes. This HA tag considerably simplifies downstream processes such as antigen and cDNA library screening, studying intracellular localization, and many other functional genomics applications.

See also: Cell Transfection Reagents