Are there common restriction sites that can be used to excise a gene out of a Gateway® plasmid?

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Answer

The core region of the att sites contains the recognition sequence for the restriction enzyme BsrGI. Provided there are no BsrGI sites in the insert, this enzyme can be used to excise the full gene from most Gateway® plasmids. The BsrGI recognition site is 5'-TGTACA and is found in both att sites flanking the insertion site.
If a different restriction site is desired, the appropriate sequence should be incorporated into your insert by PCR.

Answer Id: E3317

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ceb20fd226a5000e4a18559916d50837_FAQ

Can I use E. coli containing an F' plasmid, such as Top 10F', to propagate a vector with the ccdB gene?

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Answer

While the F' plasmid does contain the ccdA gene that can inhibit or reduce the toxicity of the ccdB gene product, the ccdA expression level is likely to be too low, or inhibition may not be complete, and the bacteria would still be exposed to the ccdB gene product and thus not grow. Therefore, bacterial strains containing the F' plasmid are not recommended as hosts for propagation of ccdB containing vectors.

For propagation of Gateway® vectors containing ccdB, we recommend the One Shot® ccdB Survival™ 2 T1R Competent Cells (A10460), which were specifically designed for that purpose. However, please note that these cells are not validated for propagation of other ccdB-containing vectors like the older pZErO® plasmids, and in most cases they are not expected to work due to very high levels of ccdB protein expressed in those vectors.

Answer Id: E3870

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5dc420b2cb29f110092134fd15430ea0_FAQ

Can N-terminal or C-terminal tags be attached to a Gateway® Entry clone?

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Answer

To have an N-terminal tag, the gene of interest must be in the correct reading frame when using non-TOPO® adapted Gateway® entry vectors. All TOPO® adapted Gateway® Entry vectors will automatically put the insert into the correct reading frame, and to add the N-terminal tag you simply recombine with a destination vector that has N-terminal tag.

To attach a C-terminal tag to your gene of interest, the insert must lack its stop codon, and be in the correct reading frame for compatibility with our C-terminal tagged destination vectors. Again, TOPO® adapted Gateway® Entry vectors will automatically put the insert into the correct reading frame. If you do not want the C-terminal tag to be expressed, simply include a stop codon at the end of the insert that is in frame with the initial ATG.

Generally, you need to choose a destination vector before you design and clone your insert into the Entry vector. This will determine whether you need to include an initiating ATG or stop codon with your insert.

Answer Id: E3950

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9fd12edd34b7e795629873843f6868a0_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?

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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

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

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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

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af510a4ec03aced844ef5cd3277b4b3d_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?

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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

Do you offer Gateway® vectors for expression in plants?

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Answer

We do not offer any Gateway® vectors for expression in plants.

Answer Id: E9854

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f35a2a2fda93397e37ba9cec45315b52_FAQ

Can I purchase the 5X LR Clonase® buffer or 5X BP Clonase® buffer separately?

Product FAQ

Answer

We do not offer the 5X LR Clonase® buffer and 5X BP Clonase® buffer as standalone products. They are available as part of the enzyme kits.

Answer Id: E9855

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0dd65ff7d2983c8ab717067f11025f11_FAQ

How can I move my gene of interest from a Gateway®-adapted expression clone to a new Destination vector as I have lost the entry clone?

Product FAQ

Answer

We would recommend performing a BP reaction with a Donor vector in order to obtain an entry clone. This entry clone can then be used in an LR reaction with the Destination vector to obtain the new expression clone.

Answer Id: E9856

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919b5529a674f6d2956edaddb7a15612_FAQ

Do you have a recommended single-step protocol for BP/LR recombination?

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Answer

Yes, we have come up with a single-step protocol for BP/LR Clonase® reaction (http://www.lifetechnologies.com/us/en/home/life-science/cloning/gateway-cloning.html#1), where DNA fragments can be cloned into Destination vectors in a single step reaction, allowing you to save time and money.

Answer Id: E9857

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80619757f4a4dae3ad303e4aa46318b5_FAQ

Can I perform the single-step protocol for the BP/LR Clonase® reaction using BP Clonase® enzyme and LR Clonase® enzyme instead of BP Clonase® II enzyme and LR Clonase® II enzyme?

Product FAQ

Answer

In the single-step protocol for the BP/LR Clonase® reaction, we would not recommend substituting the BP Clonase® II/LR Clonase® II enzymes with BP Clonase® /LR Clonase® enzymes as this would result in very low recombination efficiency.

Answer Id: E9858

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40248bad60307808063848f3acac9030_FAQ