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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Can I sequence a peptide that is acetylated or biotinylated?

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No. These groups effectively block the N terminus.

Answer Id: E1267

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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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What size targets can be amplified by the SuperScript™ One-Step RT-PCR Systems?

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The SuperScript™ III One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is recommended for amplifying RNA targets up to 4.5 kb and the SuperScript™ II One-Step RT-PCR System with Platinum™ Taq DNA Polymerase is recommended for amplifying RNA targets up to 3.5 kb. Even though the SuperScript™ II and III One-Step RT-PCR Systems with Platinum™ Taq High Fidelity can amplify smaller targets, it is recommended for amplifying RNA targets from 1 kb up to 9 kb.

Answer Id: E3213

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Will the Anti-HisG antibody recognize proteins made in pDEST17 (N-terminal His tag vector)?

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Answer

No, the Anti-HisG antibody detects the N-terminal polyhistidine (6x His) tag followed by glycine: HHHHHHG. The proteins made from the pDEST17 vectors do not have this glycine following the His tag and therefore will not be recognized by this antibody.

Answer Id: E3881

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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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Where is the ATG relative to the 5' attB site in a Gateway™ expression clone?

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This depends on whether you are expressing a fusion or a native protein in the Gateway™ destination vector. For an N-terminal fusion protein the ATG will be given by the destination vector and it will be upstream of the attB1 site. For a C-terminal fusion protein or a native protein, the ATG should be provided by your gene of interest, and it will be downstream of the attB1 site.

Answer Id: E3202

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From where does Gateway™ get its lambda nomenclature, and is it consistent with textbook nomenclature for lambda recombination?

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Answer

The Gateway™ nomenclature is consistent with lambda nomenclature, but we use numbers to differentiate between modified versions of the att sites (attB1, attB2, attP1, attP2, and so on). We have introduced mutations in the att sites to provide specificity and directionality to the recombination reaction. For example, attB1 will only recombine with attP1 and not with attP2.

Answer Id: E3209

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How does adding Platinum™ Taq DNA Polymerase improve SuperScript™ One-Step RT-PCR performance?

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Platinum™ Taq DNA Polymerase is precomplexed with a mixture of antibodies that inhibit polymerase activity until the initial denaturation step in PCR. As a result, nonspecific polymerase acitivty at lower temperatures during set-up and reverse transcription is eliminated, which provides greater yield and specificity of intended product.

Answer Id: E3214

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Do the Gateway vectors pDEST14, pDEST15, pDEST17, and pDEST24 contain transcription terminators?

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Answer

Yes, all T7 DEST Vectors carry T7 transcription termination sequences downstream of the attR2 site.

Answer Id: E3900

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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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What is the purpose of the Proteinase K step following a Gateway™ LR Recombination reaction, and is it critical to the results?

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Answer

When the LR reaction is complete, the reaction is stopped with Proteinase K and transformed into E. coli resulting in an expression clone containing a gene of interest. A typical LR reaction followed by Proteinase K treatment yields about 35,000 to 150,000 colonies per 20ul reaction. Without the Proteinase K treatment, up to a 10 fold reduction in the number of colonies can be observed. Despite this reduction, there are often still enough colonies containing the gene of interest to proceed with your experiment, so the Proteinase K step can be left out after the LR reaction is complete if necessary.

Answer Id: E4230

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Will Gateway™ att sites affect the expression of my protein?

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Answer

Expression experiments have shown that the extra amino acids contributed by the attB site to a fusion protein will most likely have no effect on protein expression levels or stability. In addition, they do not appear to have any effect on two-hybrid interactions in yeast. However, as is true with the addition of any extra sequences that result from tags, the possible effects will be protein-dependent.

Answer Id: E3200

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What is the smallest fragment that can be used in a Gateway™ reaction?

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Answer

The smallest size we have recombined is a 70 bp piece of DNA located between the att sites. Very small pieces are difficult to clone since they negatively influence the topology of the recombination reaction.

Answer Id: E3198

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How would you incorporate a leader sequence for secretion into an entry vector?

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Answer

A simple way to express a protein with a leader sequence is to have the leader sequence encoded in the destination vector. The other option is to have the leader sequence subcloned into the entry vector using restriction enzymes, or incorporate the leader sequence into the forward PCR primer when cloning a PCR product into the entry vector. Please see Esposito et al. (2005), Prot. Exp. & Purif. 40, 424-428 for an example of how a partial leader sequence for secretion was incorporated into an entry vector.

Answer Id: E3203

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