The Carboxy Flexi® Enzyme Blend (SgfI & EcoICRI) is required to cut acceptor Flexi® Vectors containing a carboxy-terminal peptide or protein tag. The donor vector (i.e., the clone containing the protein coding region of interest) is cut with SgfI and PmeI in a separate reaction. The two restriction digests are heat-inactivated and then combined with ligase to transfer the protein coding region to the acceptor vector, thereby appending the carboxy-terminal tag to the protein of interest. The ligated product no longer contains the EcoICRI site of the acceptor vector or the PmeI site of protein coding region, as these two blunt-end cutter target sites are not recreated in the ligation. Since the acceptor Flexi® Vector contains a lethal barnase gene, complete digestion with Carboxy Flexi® Enzyme Blend is not required for good transfer rates of the protein coding region from the donor Flexi® Vector.
SgfI recognition site:
EcoICRI recognition site:
5X Flexi® Digest Buffer: The composition of 5X Flexi® Digest Buffer is 50mM Tris-HCl (pH 7.9 at 37°C), 50mM MgCl2, 250mM NaCl, 5mM DTT, 0.5mg/ml acetylated BSA.
Storage Buffer: The Carboxy Flexi® Enzyme Blend (SgfI & EcoICRI) is supplied in 10mM Tris-HCl (pH 7.3 at 25°C), 150mM KCl, 25mM NaCl, 0.1mM EDTA, 1mM DTT, 0.5mg/ml BSA, 0.05% Thesit® and 50% glycerol.