I see that the pSectag2 vectors carry the Zeocin™ antibiotic-resistance gene driven by the EM7 promoter. Can I use Zeocin™ antibiotic for selection when I propagate these vectors in E. coli?

Product FAQ

Answer

Yes, you can use Zeocin™ antibiotic for selection in E. coli. However, keep in mind that for Zeocin™ antibiotic to be active, the salt concentration of the medium must remain low (<90 mM) and the pH must be 7.5. Prepare LB broth and LB agar plates using low-salt (5 g NaCl/liter) LB.

Answer Id: E9212

Was this answer helpful?

Yes
No
Thank you for your response

Can I propagate my pSecTag2 vector in E. coli containing the Tn5 gene?

Product FAQ

Answer

pSectag2 vectors have the Zeocin™ antibiotic-resistance marker for selection in E. coli, and any E. coli strain that contains the complete Tn5 transposable element (i.e., DH5alphaF’IQ, SURE, SURE2) encodes the ble (bleomycin) resistance gene that confers resistance to the Zeocin™ antibiotic. Hence, for the most efficient selection, we highly recommend choosing an E. coli strain that does not contain the Tn5 gene.

Answer Id: E9213

Was this answer helpful?

Yes
No
Thank you for your response

What is the difference between pZeoSV2 (+) and (-) vectors?

Product FAQ

Answer

The (+) and (-) designations refer to the orientation of the multiple cloning sites in these vectors. The vector is offered in two orientations to allow flexibility in cloning.

Answer Id: E9214

Was this answer helpful?

Yes
No
Thank you for your response

What is the advantage of using pShooter™ vectors?

Product FAQ

Answer

pShooter™ vectors are designed to localize recombinant proteins to intracellular compartments, such as the nucleus (pEF/myc/nuc and pCMV/myc/nuc), mitochondria (pCMV/myc/mito), endoplasmic reticulum (pCMV/myc/ER), or cytoplasm (pCMV/myc/cyto).

Answer Id: E9215

Was this answer helpful?

Yes
No
Thank you for your response

How soon after thawing can I use my Expi293F™cells and for how long are they good to use?

Product FAQ

Answer

Expi293F™ cells must be recovered from freezing and passaged at least 3 times using the procedure outlined in the manual to ensure optimal performance. Cells should maintain performance for at least 30 passages if maintained in accordance with the protocol in the manual.

Answer Id: E9232

Was this answer helpful?

Yes
No
Thank you for your response

Are proteins expressed from pCMV/myc/ER targeted to the lumen, membrane, or cytoplasmic surface of the ER?

Product FAQ

Answer

The ER-targeting signal in pCMV/myc/ER vector consists of an N-terminal signal peptide to direct the protein into the secretory compartment and a C-terminal peptide (SEKDEL) to retain the protein in the ER. This results in the protein being retained in the ER lumen by binding to a receptor. The original reference for this vector is: Munro S and Pelham HRB (1987) A C-Terminal Signal Prevents Secretion of Luminal ER Proteins. Cell 48:899-907.

Answer Id: E9216

Was this answer helpful?

Yes
No
Thank you for your response

If I let my Expi293F™ cells grow to a higher density than recommended, will they be okay?

Product FAQ

Answer

While the Expi293™ Expression Medium can support much higher cell densities, we do not recommend growing your Expi293F™ cultures beyond 5-6 x 10e6 cells/mL, as subsequent transfection and protein expression efficiencies may be reduced. At higher densities, there is also the increased possibility of reaching the point of reduced culture viability. If your seed culture does exceed 5-6 x 10e6 cells/mL, passage them once or twice as detailed in the manual, monitoring them for viability and growth rate. Perform a test transfection using the Protein Expression Control IgG or an expression construct of known yield to determine if cell expression performance has been impacted.

Answer Id: E9233

Was this answer helpful?

Yes
No
Thank you for your response

I would like to create a stable algae line via non-random integration. Which kit would you recommend?

Product FAQ

Answer

We recommend our Synechococcus kits, where the integration is directed to the Neutral Site 1 of the algae genome.

Answer Id: E9573

Was this answer helpful?

Yes
No
Thank you for your response

Where in the mitochondria would my protein be targeted to with pCMV/myc/mito vector?

Product FAQ

Answer

In the pCMV/myc/mito vector, the mitochondrial targeting signal is an N-terminal signal sequence (presequence of the subunit VIII of human cytochrome C oxidase), which lets the fusion go through the mitochondrial membrane. The signal sequence is cleaved off so the fusion is inside the mitochondria (but also may be associated with the inner membrane).

Answer Id: E9217

Was this answer helpful?

Yes
No
Thank you for your response

How many days should I wait before harvesting my protein in the Expi293™ Expression System?

Product FAQ

Answer

The optimal expression time will be different for each protein, but most tested proteins fall within a 3-7 day window. As the system scales well, it is recommended you run a small-scale pilot experiment to determine when to harvest your protein of interest prior to scaling up.

Answer Id: E9234

Was this answer helpful?

Yes
No
Thank you for your response

What is the shelf life/competency of the Chlamydomonas cells?

Product FAQ

Answer

After 6 months of storage, the Chlamydomonas cells did not lose their competency. It is expected that they may with longer storage times, but we have yet to gather data points for loss of competency after one year of storage.

Answer Id: E9574

Was this answer helpful?

Yes
No
Thank you for your response

How do your episomal mammalian expression vectors bring about episomal replication? Can I use these vectors in any cell line?

Product FAQ

Answer

Our episomal mammalian expression vectors (pCEP4, pREP4, and pEBNA-DEST) contain the Epstein Barr Virus (EBV) origin of replication (oriP) and the Epstein-Barr nuclear antigen (EBNA-1) for high-copy, transient, or stable episomal replication in human, primate, canine, and porcine cell lines. They do not bring about episomal expression in murine or rodent cell lines. pEBNA-DEST may also be used with stem cells.

Answer Id: E9218

Was this answer helpful?

Yes
No
Thank you for your response

Is there a positive control included with the Expi293™ MembranePro™ Expression System?

Product FAQ

Answer

No. However, you can visually tell if MembranePro™ particles formed via a pellet at the bottom of the tube following precipitation. You can also test the function of your MembranePro™ particles via receptor-ligand binding studies.

Answer Id: E9252

Was this answer helpful?

Yes
No
Thank you for your response

Can I leave my Expi293F™ culture expressing for more than 7 days?

Product FAQ

Answer

The growth and expression characteristics of Expi293F™ cells are such that, by 7 days post-transfection, the culture medium should be close to being spent and maximal protein expression should have already been achieved. Continued incubation will result in a large decrease in cell culture viability.

Answer Id: E9235

Was this answer helpful?

Yes
No
Thank you for your response

How do I upgrade from Vector NTI Advance™ v10 License to v11 License?

Product FAQ

Answer

Version 11 is fully backward-compatible with data stored in v10 (and in v9 and v8). Full details of how to upgrade from v10 to v11 are in the v11 Installation and Licensing Guide. Briefly:
•First, back up your existing v10 Local Database to a secure location.
•Uninstall v10 using the Add/Remove Programs functionality in your Windows™ Control Panel.
•Download and install v11 from our Downloads Page, or install v11 from the Vector NTI™ Advance 11 CD.
•During installation, you will be able to point the v11 software to the saved copy of your Local Database, and all of your existing data will be imported into the redesigned v11 Local Database automatically.

Answer Id: E5354

Was this answer helpful?

Yes
No
Thank you for your response