What is Gateway® Cloning Technology?
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: 3029
What primer purity should be used for adding attB sites to my PCR product?
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: 3172
What is the smallest fragment that can be used in a Gateway® reaction?
Can the attB primers anneal in a non-specific manner?
Will Gateway® att sites affect the expression of my protein?
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: 3200
Are the Gateway® attB1 and attB2 sites the same as the attB site used for recombination into E. coli by bacteriophage lambda?
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: 3201
Where is the ATG relative to the 5' attB site in a Gateway® expression clone?
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: 3202
How would you incorporate a leader sequence for secretion into an entry vector?
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: 3203
How clean must my DNA be to use in a Gateway® cloning reaction?
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: 3204
How many times can I thaw BP Clonase® II and LR Clonase® II?
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: 3205
Will increasing the Gateway® cloning reaction time improve recombination efficiency?
What are the prerequisites for Gateway® cloning and expression?
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: 3207
What is the first step in an experiment with the Gateway® system?
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: 3208
From where does Gateway® get its lambda nomenclature, and is it consistent with textbook nomenclature for lambda recombination?
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: 3209
Where can I get Gateway® vector sequences and maps?
Vector information can be found in the product manuals or directly on our web site by entering the catalog number of the product in the search box. The vector map, cloning site diagram, and sequence information will be linked to the product page.
Answer Id: 3210