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.thermofisher.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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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?

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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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Can I use doxycycline instead of tetracycline as an inducer in the T-REx™ system?

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Doxycycline may be used as an alternative inducing agent in the T-REx™ system. It is similar to tetracycline in its mechanism of action, and exhibits similar dose-response and induction characteristics as tetracycline in the T-REx™ system. Doxycycline has been shown to have a longer half-life than tetracycline (48 hours vs. 24 hours, respectively). We do not offer doxycycline, but it may be obtained from Sigma (Cat. No. D9891).

Answer Id: E9163

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I am interested in a mammalian expression system where I can have regulated expression of my gene of interest. I see that you offer multiple systems for this purpose. Can you describe the main features of each system?

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Answer

We offer three unique mammalian expression systems for inducible/regulated expression of the gene of interest:

- T-REx™ system
- Flp-In™ T-REx™ system
- GeneSwitch™ system

Please see below to see how they compare with one another:
System -- Basal Expression Level -- Induced Expression Level -- Response time to Maximal Expression -- Transgenic Appliation
T-Rex&trade system -- Low -- Highest -- High -- Suitable
Flp-In™ T-REx™ system -- Lower -- High -- 24-48 hrs -- Suitable
GeneSwitch™ system -- Lowest -- High -- 24-48 hrs -- Suitable

Answer Id: E9159

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What is the advantage of the Flp-In™ T-REx™ system over the T-REx™ system?

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The Flp-In™ T-REx™ system combines the targeted integration offered by the Flp-In™ system with the powerful inducible expression offered by the T-REx™ system. It allows generation of isogenic, inducible, stable cell lines and permits polyclonal selection of these cell lines. Once the Flp-In™ T-REx™ host cell line containing an integrated FRT site has been created, subsequent generation of Flp-In™ T-REx™ cell lines expressing the gene(s) of interest is rapid and efficient.

Answer Id: E9164

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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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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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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?

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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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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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Can the Clontech Tet-On™ system or Tet-Off™ system components be used with your T-REx™ tetracycline-regulated mammalian expression system?

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Answer

No. The two systems are not compatible since they utilize different strategies for promoter regulation. The T-REx™ system is designed such that native E. coli tet-repressor protein molecules bind to specific tet-operator sequences (2X TO) just downstream of the TATA box in the full length CMV promoter in the expression vector. This binding keeps the promoter silent simply by preventing the normal transcription machinery from productive assembly at the TATA box. Incidentally, it is this full length CMV promoter region that permits higher induced expression levels relative to other systems.

The recombinant ’repressor’ proteins utilized in Clontech’s system are actually recombinant fusion proteins which also contain a potent transcriptional transactivator. The Clontech system places operator sequences 5’ to the TATA box and relies upon the VP16 transactivator to promote transcription. These repressor-transactivator fusion constructs would have unpredictable and unreliable effects at the CMV promoter in our expression constructs. Additionally, the tet-repressor protein produced from the pCDNA™6/TR construct in the T-REx™ system has no transactivation domain and so would exert little regulatory effect at the minimal promoter region (non-full length CMV) found in the Clontech response plasmids.

Answer Id: E3947

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

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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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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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Can I use the anti-HisG antibody for western detection of the His tag in Gateway™ pT-REx™ DEST31?

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Answer

The Gateway™ pT-REx™ DEST31 vector contains an N-terminal 6xHis tag (the 6XHis in this vector is not followed by Glycine (G) or -COOH). Hence, it cannot be detected using anti-HisG or anti-His (C-terminal) antibodies. Instead, we recommend using an anti-6xHis antibody (Cat. No. 372900).

Answer Id: E9161

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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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Why is sequential transfection recommended over co-transfection in the T-REx™ and GeneSwitch™ systems?

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Answer

When a co-transfection is performed, there is no way of testing the double stable cell line for functional TetR or GeneSwitch™ protein, respectively. On the other hand, when sequential transfection is performed, one can functionally test the generated T-REx™ or GeneSwitch™ cell line by transiently transfecting the lacZ expression control plasmid and then picking a clone that shows the lowest basal level of expression of lacZ in the absence of the inducer, and the highest level of lacZ in the presence of the inducer. This clone can then be expanded and used to transfect the T-REx™ or GeneSwitch™ expression construct, as the case may be.

Answer Id: E9167

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