Protocols

DNA Cloning:

The customer insert either provided by customer or from our DNA databank is amplified first via PCR technology with high fidelity DNA polymerase.  The amplified fragment is inserted into plasmids pET28a with the corresponding restriction enzyme digestion.  All clones will be confirmed with either PCR assay or partial DNA sequencing.

Protein Expression/Purification:

All expression vectors are transformed into E. coli strain BL21 (DE) or BL21(DE3)pLysS.  Protein is induced with addition of IPTG for several hours. Cell lysis is carried out by sonication and cell debris removed by centrifugation.  Supernatant flows through the Ni-NTA resin column.  Protein is eluted from the column by solution containing different concentrations of imidazole.

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