pcDNA.6/myc-His A, B, and C are 5.1-kb vectors designed for overproduction of recombinant proteins in mammalian cell lines. High-level stable and transient expression can be carried out in most mammalian cells. pcDNA.6/myc-His载体含有以下元件: Human cytomegalovirus immediate-early (CMV) promoter for high-level expression in a wide range of mammalian cells.
Three reading frames to facilitate in-frame cloning with a C-terminal peptide encoding the myc (c-myc) epitope and a polyhistidine (6xHis) metal-binding tag.
Blasticidin resistance gene (bsd) for selection of stable cell lines (Kimura et al.,1994).
Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen (e.g., COS7).
The control plasmid, pcDNA.6/myc-His/lacZ, is included for use as a positive control for transfection, expression, and detection in the cell line of choice. 实验流程
使用pcDNA6/myc-His 载体克隆和表达目的基因的实验流程如下： Consult the multiple cloning sites described on pages 3–5 to determine which vector (A, B, or C) should be used to clone your gene in-frame with the C-terminal myc epitope and the polyhistidine tag. Ligate your insert into the appropriate vector and transform into E. coli. Select transformants on 50–100 μg/ml ampicillin or 50 μg/ml blasticidin.
Analyze your transformants for the presence of the insert by restriction digestion.
Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in-frame with the C-terminal peptide.
Transfect your construct into the cell line of choice using your own method of transfection.
Test for expression of your recombinant gene by western blot analysis or functional assay. For antibodies to the myc epitope or the C-terminal polyhistidine tag, see the next page.
To purify your recombinant protein, you may use metal-chelating resin such as ProBond. ProBond. resin is available separately.
