Can I sequence a peptide that is acetylated or biotinylated?

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Answer

No. These groups effectively block the N terminus.

Answer Id: E1267

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9938e76f3b572f7bc6c61808f1015d8a_FAQ

What is Gateway® Cloning Technology?

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Answer

Gateway® Cloning Technology is an easy-to-use system for cloning and subcloning DNA segments (e.g. genes of interest), facilitating gene functional analysis, protein expression, and the integration of technology platforms. One can also readily clone PCR products into so-called Gateway® "Entry" vectors. To shuttle inserts from one vector to another, the Gateway® Cloning Technology uses bacteriophage lambda-based site-specific recombination. There is no need to use restriction enzymes and ligase to subclone inserts.

One advantage of Gateway® Cloning Technology is that genes present in a single Gateway® Entry vector can be subcloned into multiple different Gateway® Destination vectors. After this 1 hour in vitro subcloning reaction, a high percentage of the colonies obtained carry the desired expression clone. For more details, please see the product manual for cat# 12535029 or cat# 12535037.

Answer Id: E3029

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ad3dd87d7339c6d0528671c7979f0bf5_FAQ

What primer purity should be used for adding attB sites to my PCR product?

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Answer

Standard desalted purity is generally sufficient for creating attB primers. We examined HPLC-purified oligos for Gateway® cloning (about 50bp long) and found only about a 2-fold increase in colony number over standard desalted primers. If too few colonies are obtained, you may try to increase the amount of PCR product used and/or incubate the BP reaction overnight.

Answer Id: E3172

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fc138a83fd2868b7715e394b57dbb0f5_FAQ

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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f8a0862ae4d4cb5227ebf626536c1543_FAQ

Can the attB primers anneal in a non-specific manner?

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Answer

No, attB primers are highly specific under standard PCR conditions. We have amplified from RNA (RT-PCR), cDNA libraries, genomic DNA, and plasmid templates without any specificity problems.

Answer Id: E3199

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920a2bb87d560a097ed05e08770d4ad8_FAQ

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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9f3eff742eb70470c411d2279b318f04_FAQ

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

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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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2e1ceb9dc9a8f7e7dab392a49e44027b_FAQ

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

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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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b2136e9101930517ff80aa2b26299686_FAQ

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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38cf46385cbdf0172f133d889168178a_FAQ

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

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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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56487e9004b86b2977416b62b7127e37_FAQ

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

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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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ee1e88dfa692a2bf77287090d02682f2_FAQ

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

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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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554d9002c940a0798ebd68824b1cceea_FAQ

What are the prerequisites for Gateway® cloning and expression?

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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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ad45619d4aac6530f2f50ae03199dcac_FAQ

What is the first step in an experiment with the Gateway® system?

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Answer

The first step is to create an Entry clone for your gene of interest. We have 3 options to do this: The first is by BP recombination reaction using the PCR Cloning System with Gateway® Technology. This is recommended for cloning large (>5 kb) PCR products. We also have Gateway® compatible TOPO® Cloning vectors such as pCR®8/GW/TOPO® and pENTR™/D-TOPO®. The final option is to use restriction enzymes to clone into a pENTR™ Dual Selection vector.

Answer Id: E3208

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f59f8b3b541fa4d94f856ce143b33bf5_FAQ

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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b0122205db90396a3568026adc1afa18_FAQ