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The PinPoint Xa Protein Purification System is designed for the production and purification of fusion proteins that are biotinylated in vivo. The DNA coding for the protein of interest is cloned into a PinPoint Vector downstream of a sequence encoding a peptide that becomes biotinylated in vivo. Biotinylated fusion proteins are produced in E. coli and are affinity-purified using the SoftLink Soft Release Avidin Resin. This proprietary resin allows elution of the fusion protein under nondenaturing conditions. The PinPoint Vectors feature the encoded endoproteinase Factor Xa (pronounced ten a) proteolytic site that provides a way to separate the purification tag from the native protein, and the vectors carry a convenient multiple cloning region for ease in construction of fusion proteins. The system contains vectors in all possible sense reading frames, an avidin-conjugated resin, Streptavidin-Alkaline Phosphatase, a purification column and biotin. The PinPoint Xa Control Vector contains the chloramphenicol acetyltransferase (CAT) gene and is provided as a means of monitoring protein expression, purification and processing conditions. The system generally yields 1-5mg of protein per liter of culture.

The pGEM-9Zf(-) Vector is a recombinant plasmid designed to provide a versatile range of cloning strategies, efficient synthesis of RNA in vitro and the production of single-stranded DNA. The plasmid contains T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the alpha-peptide coding region of beta-galactosidase. Insertional inactivation of the alpha-peptide allows recombinant clones to be identified directly by color screening on indicator plates when using appropriate E. coli strains (e.g., JM109). The multiple cloning region is unique and includes restriction sites for NsiI, SpeI, HindIII, XbaI, EcoRI, SalI and SacI.

The pGEM-7Zf(+) and pGEM-7Zf(-) Vectors are derivatives of the pGEM-3Zf(+) Vector and contain the origin of replication of the filamentous phage f1. These plasmids serve as standard cloning vectors, as templates for in vitro transcription and can be used for the production of circular ssDNA. These plasmids contain SP6 and T7 RNA polymerase promoters flanking a region of multiple cloning sites within the alpha-peptide coding region of beta-galactosidase. Insertional inactivation of the alpha-peptide allows recombinant clones to be identified directly by color screening on indicator plates when using appropriate E. coli strains (e.g., JM109). The multiple cloning region is unique and includes restriction sites for ApaI, AatII, SphI, XbaI, XhoI, EcoRI, KpnI, SmaI, ClaI, HindIII, BamHI, SacI, BstXI and NsiI. This arrangement is designed specifically for generating unidirectional deletions with the Erase-a-Base System. pGEM-7Zf(+) and pGEM-7Zf(-) Vectors are identical except for the orientation of the f1 origin.

The pGEM-5Zf(+) Vector serves as a standard cloning vector and as a template for in vitro transcription. This vector contains T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the alpha-peptide coding region of beta-galactosidase. Insertional inactivation of the alpha-peptide allows recombinant clones to be identified directly by color screening on indicator plates when using appropriate E. coli strains (e.g., JM109). The multiple cloning region contains unique restriction sites for ApaI, AatII, SphI, NcoI, SacII, EcoRV, SpeI, NotI, PstI, SalI, NdeI, SacI, BstXI and NsiI.

The pGEM-11Zf(+) and pGEM-11Zf(-) Vectors can be used as standard cloning vectors, as templates for in vitro transcription and for the production of ssDNA. These plasmids contain T7 and SP6 RNA polymerase promoters flanking a multiple cloning region within the alpha-peptide coding region of beta-galactosidase. Insertional inactivation of the alpha-peptide allows recombinant clones to be identified directly by color screening on indicator plates when using appropriate E. coli strains (e.g., JM109). The multiple cloning region contains unique restriction sites for SfiI, SacI, EcoRI, SalI, XhoI, BamHI, ApaI, XbaI, NotI, SphI, NsiI and HindIII. The pGEM-11Zf(-) and pGEM-11Zf(+) Vectors are identical except for the orientation of the f1 origin.