I am working with a mouse cell line and would like to express my gene at high levels using one of your vectors with the CMV promoter. Do you foresee any problems with this approach?

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Answer

The CMV promoter is known to be downregulated over time in mouse cell lines. Hence, we recommend using one of our non-CMV vectors, such as those with the EF1alpha or UbC promoter, for long-term expression in mouse cell lines.

Answer Id: E9152

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aa6000854a733631ad122a7c944854d0_FAQ

Do you offer a GFP-expressing mammalian expression vector that I can use as a control to monitor my transfection and expression?

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Answer

We offer pJTI™ R4 Exp CMV EmGFP pA Vector, Cat. No. A14146, which you can use to monitor your transfection and expression.

Answer Id: E9154

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2c85f53110326245a2efcdaf9c503be0_FAQ

Is it okay if my construct has an ATG that is upstream of the ATG in my gene of interest? Will it interfere with translation of my gene?

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Answer

Translation initiation will occur at the first ATG encountered by the ribosome, although in the absence of a Kozak sequence, initiation will be relatively weak. Any insert downstream would express a fusion protein if it is in frame with this initial ATG, but levels of expressed protein are predicted to be low if there is a non-Kozak consensus sequence. If the vector contains a non-Kozak consensus ATG, we recommend that you clone your gene upstream of that ATG and include a Kozak sequence for optimal expression.

Answer Id: E9180

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16c54268c5857e0549e9da663fb0c2ef_FAQ

I am using a mammalian expression vector that has the neomycin resistance gene. Can I use neomycin for stable selection in mammalian cells?

Product FAQ

Answer

No; neomycin is toxic to mammalian cells. We recommend using Geneticin® (a.k.a. G418 Sulfate), as it is a less toxic and very effective alternative for selection in mammalian cells.

Answer Id: E9181

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86410c89d41682a89d98002342b5c3f1_FAQ

I used a mammalian expression vector but do not get any expression of my protein. Can you help me troubleshoot?

Product FAQ

Answer

Here are possible causes and solutions:

- Try the control expression that is included in the kit
Possible detection problem:

- Detection of expressed protein may not be possible in a transient transfection, since the transfection efficiency may be too low for detection by methods that assess the entire transfected population. We recommend optimizing the transfection efficiency, doing stable selection, or using methods that permit examination of individual cells. You can also increase the level of expression by changing the promoter or cell type.
- Expression within the cell may be too low for the chosen detection method. We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot. Protein might be degraded or truncated: Check on a Northern. Possible time-course issue: Since the expression of a protein over time will depend upon the nature of the protein, we always recommend doing a time course for expression. A pilot time-course assay will help to determine the optimal window for expression. Possible cloning issues: Verify clones by restriction digestion and/or sequencing.

Answer Id: E9182

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78bbc5bb321b11c2f6a40a2e143aae2a_FAQ

I performed stable selection but my antibiotic-resistant clones do not express my gene of interest. What could have gone wrong?

Product FAQ

Answer

Here are possible causes and solutions:

Detection method may not be appropriate or sensitive enough:
- We recommend optimizing the detection protocol or finding more sensitive methods. If the protein is being detected by Coomassie/silver staining, we recommend doing a western blot for increased sensitivity. The presence of endogenous proteins in the lysate may obscure the protein of interest in a Coomassie/silver stain. If available, we recommend using a positive control for the western blot.
- Insufficient number of clones screened: Screen at least 20 clones.
- Inappropriate antibiotic concentration used for stable selection: Make sure the antibiotic kill curve was performed correctly. Since the potency of a given antibiotic depends upon cell type, serum, medium, and culture technique, the dose must be determined each time a stable selection is performed. Even the stable cell lines we offer may be more or less sensitive to the dose we recommend if the medium or serum is significantly different.
- Expression of gene product (even low level) may not be compatible with growth of the cell line: Use an inducible expression system.
- Negative clones may result from preferential linearization at a vector site critical for expression of the gene of interest: Linearize the vector at a site that is not critical for expression, such as within the bacterial resistance marker.

Answer Id: E9183

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559c6028788610ad1cbc4bc184da429d_FAQ

How do your Vivid Colors™ fluorescent protein vectors compare to the previously sold Clontech BD Living Colors™ fluorescent protein vectors in terms of overall brightness?

Product FAQ

Answer

In addition to the key mutations that enhanced the brightness of the Clontech fluorescent proteins, we have added further genetic enhancements to the fluorescent proteins to increase the quantum yield. Side-by-side comparisons have shown the fluorescence intensity of our Vivid Colors™ fluorescent protein expression vectors to be at least equivalent (or better than) the comparable Clontech BD Living Colors™ fluorescent protein expression vectors. 

Answer Id: E9190

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aca6b39847c35d2305282e7e1a24f3f4_FAQ

How does Cycle 3 GFP compare with EmGFP and EGFP?

Product FAQ

Answer

EmGFP is the next-generation variant of EGFP, and it has been further optimized for mammalian expression. Both EmGFP and EGFP can be visualized using the same filter sets (FITC) and settings. When used with the recommended filter sets and settings, Cycle 3 GFP is as bright as EGFP or EmGFP. However, when used with FITC filter sets and settings, Cycle 3 GFP is not as bright as EmGFP or EGFP.

- Excitation/emission maxima for EGFP: 488 nm/507-509 nm
- Excitation/emission maxima for EmGFP: 487 nm/509 nm
- Excitation/emission maxima for Cycle 3 GFP: 395 nm (primary) and 478 nm (secondary)/507 nm

Answer Id: E9191

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8cd28cea4c7d9e5374221677f31b5a93_FAQ

Are the fluorescent proteins you offer (EmGFP, YFP, CFP, BFP, and Cycle 3 GFP) humanized?

Product FAQ

Answer

Yes, all of the fluorescent proteins offered by us (EmGFP, YFP, CFP, BFP, and Cycle 3 GFP) have been humanized for optimal mammalian expression.

Answer Id: E9192

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8ea7cea80a3d77432e295863bfc670c3_FAQ

What are the excitation and emission maxima for your fluorescent proteins (EmGFP, YFP, BFP, CFP, and Cycle 3 GFP)?

Product FAQ

Answer

Excitation and emission maxima for our fluorescent proteins are as follows:
- EmGFP: Excitation: 487 nm; Emission: 509 nm
- YFP: Excitation: 514 nm; Emission: 527 nm
- BFP: Excitation: 308-383 nm; Emission: 440-447 nm
- CFP: Excitation: 452 nm; Emission: 505 nm
- Cycle 3 GFP: Primary excitation: 395 nm; Secondary Excitation: 478 nm; Emission: 507 nm

Answer Id: E9193

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389b07c40574f80757a29a47523c47eb_FAQ

What are the recommended filter sets for detection of EmGFP, YFP, CFP, and BFP by fluorescence microscopy?

Product FAQ

Answer

EmGFP, YFP, CFP, and BFP can be detected using standard FITC filter sets and settings. However, for optimal detection of the fluorescence signal, filter sets optimized for detection within the excitation and emission ranges for each fluorescent protein are recommended. The recommended filter sets are as follows: EmGFP: Omega filter set XF100 YFP: Omega filter set XF1042 Chroma filter set 41028 CFP: Omega filter set XF114 Chroma filter set 31044 BFP: Omega filter set XF10 Chroma filter set 31021 For information on obtaining filter sets, please contact Omega Optical, Inc. (www.omegafilters.com) or Chroma Technology Corporation (www.chroma.com) directly.

Answer Id: E9194

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66243f1f7a99ec3770af7fd828f4fadf_FAQ

Can GFP fluorescence be detected in cells that have been stained for beta-galactosidase?

Product FAQ

Answer

We recommend looking for GFP fluorescence before staining for beta-galactosidase. This is because the beta-galactosidase staining process produces a very high autofluorescence that will interfere with detection of GFP fluorescence.

Answer Id: E9196

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97490c530d4a125237e4e3116df00999_FAQ

Your Gateway®-adapted TOPO® vectors are supplied with a control template and control primers. Can I obtain the sequence of the control template?

Product FAQ

Answer

The sequence of the control template is proprietary.

Answer Id: E9853

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9b346edad7ff3fe049bc58978e079af0_FAQ