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Product FAQ

Are the Gateway™ attB1 and attB2 sites the same as the attB site used for recombination into E. coli by bacteriophage lambda?

Answer

The Gateway™ attB sites are derived from the bacteriophage lambda site-specific recombination, but are modified to remove stop codons and reduce secondary structure. The core regions have also been modified for specificity (i.e., attB1 will recombine with attP1 but not with attP2).

Answer Id: E3201

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Product FAQ

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

Answer

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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Product FAQ

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

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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Product FAQ

How clean must my DNA be to use in a Gateway™ cloning reaction?

Answer

Mini-prep (alkaline lysis) DNA preparations work well in Gateway™ cloning reactions. It is important that the procedure remove contaminating RNA for accurate quantification. Plasmid DNA purified with our S.N.A.P.™ nucleic acid purification kits, ChargeSwitch™ kits, or PureLink™ kits are recommended.

Answer Id: E3204

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Product FAQ

How many times can I thaw BP Clonase™ II and LR Clonase™ II?

Answer

BP Clonase™ II and LR Clonase™ II can be freeze/thawed at least 10 times without significant loss of activity. However, you may still want to aliquot the enzymes to keep freeze/thaw variability to a minimum.

These enzymes are more stable than the original BP and LR Clonase™ and can be stored at -20°C for 6 months.

Answer Id: E3205

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Product FAQ

Must PCR conditions be changed once the original PCR primers have attB sequence added to them?

Answer

Since the attB sequences are on the 5' end of oligos, they will not anneal to the target template in the first round of PCR. Sometimes the PCR product is more specific with the attB primers, probably due to the longer annealing sequence (all of attB plus gene specific sequence) after the first round of amplification. Generally there is no need to change PCR reaction conditions when primers have the additional attB sequence

Answer Id: E3222

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Product FAQ

Do you offer Gateway™ vectors for expression in plants?

Answer

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

Answer Id: E9854

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Product FAQ

Will increasing the Gateway™ cloning reaction time improve recombination efficiency?

Answer

Yes, increasing the incubation time from 1 hour to 4 hours will generally increase colony numbers 2-3 fold. An overnight incubation at room temperature will typically increase colony yield by 5-10 fold.

Answer Id: E3206

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Product FAQ

What is the influence of the attB sequence on protein function, solubility, folding, and expression?

Answer

Destination vectors that contain N-terminal fusion partners will express proteins that contain amino acids contributed from the attB1 site, which is 25 bases long. This means that in addition to any tag (6x His and/or antibody epitope tag) the N-terminus of an expressed protein will contain an additional 9 amino acids from the AttB1 sequence - the typical amino acid sequence is Thr-Ser-Leu-Tyr-Lys-Lys-Ala-Gly-nnn, where nnn will depend on the codon sequence of the insert.

Effects on protein function: A researcher (Simpson et al. EMBO Reports 11(31):287-292, 2000) demonstrated that GFP fusions (N- terminal and C-terminal) localized to the proper intracellular compartment. The expression constructs were generated using Gateway™ cloning, so the recombinant protein contained the attB1 or attB2 amino acid sequence. The localization function of the cloned recombinant proteins was preserved.

Effects on expression: We have seen no effect of the attB sites on expression levels in E. coli, insect and mammalian cells. The gus gene was cloned into bacterial expression vectors (for native and N-terminal fusion protein expression) using standard cloning techniques and expressed in bacteria. Gus was also cloned into Gateway™ Destination vectors (for native and N-terminal fusion expression) and expressed. When protein expression is compared, there was no difference in the amount of protein produced. This demonstrates that for this particular case the attB sites do not interfere with transcription or translation.

Effects on solubility: A researcher at the NCI has shown that Maltose Binding Protein fusions constructed with Gateway™ Cloning were soluble. The fusion proteins expressed had the attB amino acid sequence between the Maltose Binding Protein and the cloned protein. It is possible that some proteins containing the attB sequence could remain insoluble when expressed in E.coli.

Effects on folding: Two Hybrids screens show the same interacters identified with and without the attB sequence. Presumably correct protein folding would be required for protein-protein interactions to take place. It is possible that some proteins containing the attB sequence may not fold correctly.

Answer Id: E3223

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Product FAQ

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

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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Product FAQ

What are the prerequisites for Gateway™ cloning and expression?

Answer

The gene of interest must be flanked by the appropriate att sites, either attL (100 bp) in an Entry clone or attB (25 bp) in a PCR product. For Entry clones, everything between the attL sites will be shuttled into the Gateway™ destination vector containing attR sites, and a PCR product flanked by attB sites must be shuttled into an attP-containing donor vector such as pDONR™221.

The location of translation initiation sites, stop codons, or fusion tags for expression must be considered in your initial cloning design. For example, if your destination vector contains an N-terminal tag but does not have a C-terminal tag, the vector should already contain the appropriate translation start site but the stop codon should be included in your insert.

Answer Id: E3207

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Product FAQ

What is the difference between LR Clonase™ II and LR Clonase™ II Plus?

Answer

LR Clonase™ II Plus contains an optimized formulation of recombination enzymes for use in MultiSite Gateway™ LR reactions™. LR Clonase™ and LR Clonase™ II enzyme mixes are not recommended for MultiSite Gateway™ LR recombination reactions, but LR Clonase™ II Plus is compatible with both multi-site and single-site LR recombination reactions.

Answer Id: E5237

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Product FAQ

How large can PCR fragments be and still be cloned into a Gateway™ Entry vector?

Answer

There is no size restriction on the PCR fragments if they are cloned into a pDONR vector. The upper limit for efficient cloning into a TOPO™ adapted Gateway™ Entry vector is approximately 5 kb. A Gateway™ recombination reaction can occur between DNA fragments that are as large as 150 kb.

Answer Id: E3224

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Product FAQ

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

Answer

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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Product 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?

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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