Gateway®BP Clona II Enzyme Mix
Cat. No. 11789-020 Size: 20 reactions
Cat. No. 11789-100 Size: 100 reactions
Store at -20°C (non-frost-free freezer) Gateway® Technology
The Gateway® Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (1) to provide a rapid and highly efficient way to move DNA quences into multiple vector systems. The Gateway® Technology is schematically reprented below.
att B1-gene-att B2 u att P1-ccd B-att P2 att L1-gene-att L2 u att R1-ccd B-att R2
(expression clone) (pDONR™) (entry clone) (destination vector) The att B u att P reaction is mediated by Gateway® BP Clona II enzyme mix; the att L u att R reaction is mediated by Gateway® LR Clona II enzyme mix. ccd B is the F plasmid-encoded gene that inhibits growth of E. coli (2,3) and “gene” reprents any DNA gment of interest (e.g. PCR product, cDNA, genomic DNA). Description
Gateway® BP Clona™ II enzyme mix is a proprietary enzyme and buffer formulation containing the bacteriophage lambda recombination protein Integra (Int), the E. coli-encoded protein Integration Host Factor (IHF) (1), and reaction buffer provided in a single mix for convenient reaction t up. Gateway®BP Clona™ II enzyme mix catalyzes in vitro recombination between an att B-PCR product (or att B-containing expression clone) and an att P-containing donor vector to generate an att L-containing entry clone. Store Gateway® BP Clona™ II enzyme mix at -20ºC (non-frost-free freezer) for up to 6 months. For long-term storage, store at -80ºC.
Components Supplied20 rxns 100 rxns
P l
Gateway® BP Clona II Enzyme Mix 40 P l 200
P l
P l 200 Proteina K Solution (2 P g/P l) 40
30% PEG 8000/30 mM MgCl2 Solution 1 ml 5 ml
P l
pEXP7-tet Positive Control (50 ng/P l) 20 P l 20 Quality Control
BP Clona II enzyme mix is functionally tested in a 1 hour recombination reaction followed by a transformation assay.
Part No. 11789.II.pps Rev. Date: 22 July 2005
For rearch u only. Not intended for any animal or human therapeutic or diagnostic u.
For technical support, contact tech_
幼儿园情景剧Page 2 General Recommendations and Guidelines
x pEXP7-tet is provided for u as a positive control in the BP reaction and contains an approximately 1.4 kb fragment consisting of the tetracycline
resistance gene and its promoter flanked by att B sites.
x For att B-containing expression clones, we recommend using plasmid DNA purified with the PureLink™ HQ Mini Plasmid Purification Kit (Catalog no.
K2100-01). Mini-prep (alkaline lysis) DNA preparations are adequate for
Gateway® cloning reactions; however, in general, such DNA cannot be
quantitated by UV absorbance due to contaminating RNA and nucleotides. x You may u att B-PCR products in the BP reaction without purification. To achieve a higher percentage of desired clones, u PEG/MgCl2 precipitation (e below) to remove primer-dimers or small DNA molecules (<300 bp).
x 30% PEG 8000/30 mM MgCl2 Solution is provided to purify PCR products away from other DNA <300 bp in size, including primer-dimers. Run a 25 P l PCR reaction, dilute the PCR reaction 4-fold with TE [10 mM Tris-HCl向你介绍我
(pH 7.5-8), 1 mM EDTA], add 1/2 volume of 30% PEG 8000/30 mM MgCl2
Solution (final concentrations of 10% PEG, 10 mM MgCl2), and vortex.
Centrifuge 15 minutes at full speed in a microcentrifuge. Carefully remove
supernatant and suspend the clear pellet in TE to >10 ng/P l.
Important: U the recommended proportion of PEG/MgCl2 to ensure that
correct-sized products are removed.
x For BP reactions, the most efficient substrates are linear att B products (PCR products or expression clones) and supercoiled att P-containing donor vectors.
Supercoiled or relaxed att B substrates may be ud but will react less efficiently than linear att B substrates.
x To increa the number of colonies containing the desired entry clone, increa the incubation time from the recommended 1 hour to 4-6 hours (typically 2-3 fold more colonies) or overnight (typically 5-10 fold more colonies). Longer
incubations are recommended for genes t 5 kb to increa the yield of colonies. x We recommend using 20-50 fmol of PCR product per 10 P l reaction (where a
1 kb PCR product is ~0.65 ng/fmol). Increasing the amount of PCR product
generally yields more colonies; however, do not exceed ~250 ng of PCR
product per 10 P l reaction.
Page 3
Procedures
BP Reaction
BP Clona II enzyme mix is supplied as a 5X solution. If you wish to scale the
reaction volume, make sure the BP Clona II enzyme mix is at a final
concentration of 1X. For a positive control, u 100 ng (2 P l) of pEXP7-tet.
1. Add the following components to a 1.5 ml microcentrifuge tube at room
temperature and mix:
att B-PCR product (t10 ng/P l; final amount ~15-150 ng) 1-7 P l
P l
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Donor vector (150 ng/P l) 1 TE buffer, pH 8.0 to 8 P l
2. Thaw on ice the BP Clona II enzyme mix for about 2 minutes. Vortex the
BP Clona™ II enzyme mix briefly twice (2 conds each time).
3. To each sample (Step 1, above), add 2 P l of BP Clona II enzyme mix to the
reaction and mix well by vortexing briefly twice. Microcentrifuge briefly.
4. Return BP Clona II enzyme mix to -20q C or -80q C storage.
5. Incubate reactions at 25q C for 1 hour.
6. Add 1 P l of the Proteina K solution to each sample to terminate the
reaction. Vortex briefly. Incubate samples at 37q C for 10 minutes.
Transformation
1. Transform 1 P l of each BP reaction into 50 P l of One Shot® OmniMAX™ 2 T1
Phage-Resistant Cells (Catalog no. C8540-03). Incubate on ice for 30 minutes.
Heat-shock cells by incubating at 42q C for 30 conds. Add 250 P l of S.O.C.
Medium and incubate at 37q C for 1 hour with shaking. Plate 20 P l and 100 P l
of each transformation onto lective plates. Note: Any competent cells with
我不是完美小孩a transformation efficiency of >1.0 u 108 transformants/P g may be ud.
2. Transform 1 P l of pUC19 DNA (10 ng/ml) into 50 P l of One Shot®
OmniMAX™ 2 T1 Phage-Resistant Cells as described above. Plate 20 P l and
100 P l on LB plates containing 100 P g/ml ampicillin.
Expected Results
An efficient BP recombination reaction will produce >1500 colonies if the entire
BP reaction is transformed and plated.
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Page 4 References
1. Landy, A. (1989) Ann. Rev. Biochem. 58, 913.三五九旅
2. Bernard, P. and Couturier, M. (1992) J. Mol. Biol.226, 735.
3. Miki, T., Park, J.A., Nagao, K., Murayama, N., and Horiuchi, T. (1992) J. Mol. Biol. 225, 39. Limited U Label Licen No. 19: Gateway® Cloning Products
This product and its u is the subject of one or more of U.S. Patent Nos. 5,888,732, 6,143,557, 6,171,861, 6,270,969, 6,277,608, and 6,720,140 and/or other pending U.S. and foreign patent applications owned by Invitrogen Corporation. The purcha of this product conveys to the buyer the non-transferable right to u the purchad amount of the product and components of the product in rearch conducted by the buyer (whether the buyer is an academic or for profit entity). The purcha of this product does not convey a licen under any method claims in the foregoing patents or patent applications, or to u this product with any recombination sites other than tho purchad from Invitrogen Corporation or its authorized distributor. The right to u methods claimed in the foregoing patents or patent applications with this product for rearch purpos only can only be acquired by the u of Clona™ purchad from Invitrogen Corporation or its authorized distribu
tors. The buyer cannot modify the recombination quence(s) contained in this product for any purpo. The buyer cannot ll or otherwi transfer (a) this product, (b) its components, or (c) materials made by the employment of this product or its components to a third party or otherwi u this product or its components or materials made by the employment of this product or its components for Commercial Purpos. The buyer may transfer information or materials made through the employment of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpo, and that such collaborator agrees in writing (a) not to transfer such materials to any third party, and (b) to u such transferred materials and/or information solely for rearch and not for Commercial Purpos. Notwithstanding the preceding, any buyer who is employed in an academic or government institution may transfer materials made with this product to a third party who has a licen from Invitrogen under the patents identified above to distribute such materials. Transfer of such materials and/or information to collaborators does not convey rights to practice any methods claimed in the foregoing patents or patent applications. Commercial Purpos means any activity by a party for consideration and may include, but is not limited to: (1) u of the product or its components in manufacturing; (2) u of the product or its components to provide a rvice, information, or data; (3) u of the product or its components for therapeutic, diagnostic or prophylactic purpos; or (4) resale of the product or its components, whether or not such product or
its components are resold for u in rearch. Invitrogen Corporation will not asrt a claim against the buyer of infringement of the above patents bad upon the manufacture, u or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in rearch by the buyer in which this product or its components was employed, provided that none of (i) this product, (ii) any of its components, or (iii) a method claim of the foregoing patents, was ud in the manufacture of such product. Invitrogen Corporation will not asrt a claim against the buyer of infringement of the above patents bad upon the u of this product to manufacture a protein for sale, provided that no method claim in the above patents was ud in the manufacture of such protein. If the purchar is not willing to accept the limitations of this limited u statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a licen to u this product for purpos other than tho permitted above, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California 92008. Phone (760) 603-7200.
Limited U Label Licen No. 28: CMV Promoter
The u of the CMV promoter is covered under U.S. Patent Nos. 5,168,062 and 5,385,839 owned and licend by the University of Iowa Rearch Foundation and is sold for rearch u only. Commercial urs must obtain a licen to the patents directly from the University of Iowa Rear
五月雪黑豆加醋ch Foundation (UIRF), 214 Technology Innovation Center, Iowa City, Iowa 52242. For further information, plea contact the Associate Director of UIRF, at 319-335-4546.
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