True, science can occasionally feel like a Greek tragedy. We named our new cloning system ‘Electra’ because it allows you to select against the pMOTHER vector just as Electra destroyed her own mother Clytemnestra. Hopefully, your endeavours are more successful than those of Antigone. Thankfully, unlike the original Electra, your selection of the daughter against the mother will not require any atonement to the gods. Learn more about the classic Greek tragedy Electra: Electra story – Euripedes version Electra story – Sophocles version To PCR your ORF: we recommend you add the following ends to your primers, as these contain the Electra sites to clone directly into Electra vectors. Add 15-20 bp of your ORF to the 3′ primer end to amplify your ORF and have it compatible with any of the Electra MOTHER or Electra DAUGHTER expression vectors. Your ORF must NOT contain any SapI recognition sites, as the Electra cloning process utilizes the type IIs enzyme SapI To PCR your ORF we recommend you add the following ends to your primers, as these contain the Electra sites to clone directly into Electra vectors. Add 15-20 bp of your ORF to the 3′ primer end to amplify your ORF and have it compatible with any of the Electra MOTHER or Electra DAUGHTER expression vectors. Your ORF must NOT contain any SapI recognition sites, as the Electra cloning process utilizes the type IIs enzyme SapI Consult with an Electra Specialist today at +1 877 362 8646 or info@DNA20.com to learn more about improving your cloning and expression system. Note: the Electra Vector System reagents kit is currently available to ship only to customers in the US, Canada, Japan, and major European hubs. Electra System international shipping is available to European locations from our EU distributor, Cambridge Bioscience, Ltd. Electra System international shipping is available to Japanese locations from our distributor, Cosmo Bio Co, Ltd. From: J. H. Choi . S. Y. Lee (2003). Metabolic and Biomolecular Engineering National Research, pp 630-632. “A number of methods have been used to promote extracellular secretion of recombinant proteins from E.coli. These include the use of biochemicals, physical methods (osmotic shock, freezing and thawing), lysozyme treatment and chloroform shock. E. coli normally does not secrete proteins extracellularly except for a few classes of proteins such as toxins and hemolysin. Secreted proteins can leak from the periplasmic space into the culture medium possibly due to an increased permeability of the cell membrane during a lengthy incubation period. Small proteins secreted into the periplasm are frequently released into the culture medium (Tong et al. 2000). In general, movement of recombinant proteins from the periplasm to the culture medium is the result of compromising the integrity of the outer membrane. However, care must be exercised during such recombinant protein production so as not to compromise cellular integrity, which often causes cell death. Interestingly, glycine or Triton X-100 supplemented to the medium retarded formation of inclusion bodies in the periplasm and increased the extracellular production efficiency of recombinant proteins (Jang et al. 1999, Kaderbhai et al. 1997, Yang et al. 1998). Glycine has been found to induce morphological changes, such as an enlarged spheroidal morphology in E. coli, as it is incorporated into peptidoglycan. Glycine supplementation may slightly disrupt peptidoglycan cross-linkages and cell membrane integrity. Yang et al. (1998) reported that adding 2% (w/v) glycine dramatically increased extracellular production of sFV/TNF-α and β-glucosidase.” Source.