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Molecular Cloning is the process of producing recombinant DNA and transforming into host organisms to replicate and make more copies. Every cloning project is unique. Read More

Once you have designed the cloning scheme, gathering all of the required reagents to get you from construct to expression and analysis is not trivial. CloneWeaver helps you build a customized cloning "kit" with all of the items your cloning scheme requires. Purchase your selection instantly, save it or email it to a purchasing agent. Soon you'll have everything you require to create the construct you need.

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Vectors: PCR Cloning Change

Name Description Part Number
pTARGET Mammalian Expression Vector System Open/Close Add
Clone PCR products with T overhangs
CMV promoter for robust, consistent expression
Neomycin marker for selection of stable transfectants

The pTARGET Mammalian Expression Vector System is a convenient system for cloning PCR products and for expressing cloned PCR products in mammalian cells. The vector is prepared by digestion with EcoRV followed by addition of a 3' terminal thymidine to each end. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid in two ways. First, the overhangs prevent recircularization of the vector; second, they provide a compatible overhang for PCR products generated by thermostable polymerases that add a single deoxyadenosine, in a template-independent fashion, to the 3'-ends of amplified fragments. The pTARGET Vector also contains a modified coding sequence of the alpha-peptide of beta-galactosidase, which allows recombinants to be selected using blue/white screening. The pTARGET Vector carries the human cytomegalovirus (CMV) immediate-early enhancer/promoter region to promote constitutive expression of cloned DNA inserts in mammalian cells. This vector also contains the neomycin phosphotransferase gene, a selectable marker for mammalian cells. The pTARGET Vector can be used for transient expression or stable expression by selecting transfected cells with the antibiotic G-418.

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pGEM-T Vector Systems Open/Close Add
Insert excision with a BstZI single digest
Ligation can be completed in 1 hour at room temperature
Available with or without competent cells

The pGEM-T Vector Systems are convenient systems to clone PCR products. The pGEM-T Vector is prepared by cutting the pGEM-5Zf(+) Vector with EcoRV and adding a 3' terminal thymidine to both ends. These single 3'-T overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product into the plasmid by preventing recircularization of the vector and providing a compatible overhang for ligation of PCR products generated by thermostable polymerases that add a single deoxyadenosine, in a template-independent fashion, to the 3'-ends of amplified fragments. The multiple cloning site is flanked by recognition sites for the restriction enzyme BstZI, allowing release of the insert by a single-enzyme digestion. Alternatively, a double digestion may be used to release the insert from the vector. The pGEM-T Vector System II contains JM109 Competent Cells in addition to all of the pGEM-T Vector System I components.

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pGEM-T Easy Vector Systems Open/Close Add
Three options for removing the insert with a single digest (BstZI, EcoRI or NotI )
Ligation can be completed in 1 hour at room temperature
Available with or without competent cells

The pGEM-T Easy Vector Systems are convenient systems to clone PCR products. They offer all of the advantages of the pGEM-T Vector Systems with the added convenience of recognition sites for EcoRI and NotI flanking the insertion site. Thus, several options exist to remove the desired insert DNA with a single restriction digestion. The pGEM-T Easy Vector System II contains JM109 Competent Cells in addition to all of the pGEM-T Easy Vector System I components.

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