How do I quench GFP fluorescence?

Product FAQ

Answer

GFP fluorescence can be quenched by washing with 0.1% sodium borohydride in PBS for 30 min after fixing the cells.

Answer Id: E4929

Was this answer helpful?

Yes
No
1df06e387c930cf4fa96858e8e150036_FAQ

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?

Product FAQ

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

Was this answer helpful?

Yes
No
aa6000854a733631ad122a7c944854d0_FAQ

Which competent E. coli do you recommend using for propagation of my Gateway®-adapted mammalian Destination vector?

Product FAQ

Answer

We recommend using One Shot® ccdB Survival™ 2 T1R Competent Cells, Cat. No. A10460. This strain is resistant to the toxic effects of the ccdB gene. Note: Do not use general E. coli cloning strains, including TOP10 or DH5alpha™, for propagation and maintenance, as these strains are sensitive to ccdB effects.

Answer Id: E9153

Was this answer helpful?

Yes
No
af510a4ec03aced844ef5cd3277b4b3d_FAQ

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

Product FAQ

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

Was this answer helpful?

Yes
No
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?

Product FAQ

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

Was this answer helpful?

Yes
No
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

Was this answer helpful?

Yes
No
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

Was this answer helpful?

Yes
No
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

Was this answer helpful?

Yes
No
559c6028788610ad1cbc4bc184da429d_FAQ