pRSET/FP Vector - Thermo Fisher Scientific
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1. product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity No rights are conveyed to modify or clone the gene encoding GFP contained in this product The buyer cannot sell or otherwise 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 otherwise use this product or its components or materials made by the employment of this product or its components for Commercial Purposes 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 Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic dia gnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research Life Techno logies Corporation will not assert a claim against2. the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnost ic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that none of this product or any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies Corporation is willing to accept return of the product with a full refund For information on purchasing a license to use this product for purposes other than those permitted above contact Li censing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or outlicensing lifetech com Continued on next page 15 Purchaser Notification Continued Limited Use Label License No 267 Mutant Green Fluor escent Products Limited Use Label License No 268 Green Fluorescent Proteins Limited Use Label License No 358 Re search Use Only 16 This product and its use is the subject of one or more of U S Patent Nos 6 090 919 5 804 387 5 994 077 and foreign equival ents This product is licensed from Amersham Biosciences UK Ltd under U S Patent Nos 6 172 188 and 6 818 443 and foreign equi valents and is provided only for discovery and development of human therapeutics e g small molecules or proteins includi
3. the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose 13 Purchaser Notification Limited Use Label License No 22 Vectors and Clones En coding Histidine Hexamer Limited Use Label License No 127 GFP with Heterologous Promoter 14 This product is licensed under U S Patent Nos 5 284 933 and 5 310 663 and foreign equivalents from Hoffmann LaRoche Inc Nutley NJ and or Hoffmann LaRoche Ltd Basel Sw
4. ed for human or animal diagnostic or therapeutic uses iv Introduction Product Overview Description of the System Applications pRSET CFP pRSET EmGFP and pRSET BFP vectors are bacterial expression vectors that contain sequences encoding Fluorescent Proteins FPs FPs are derived from Green Fluorescent Protein and contain amino acid substitutions that alter the spectral properties of the proteins see page 3 for details Upon excitation these FPs emit a fluorescent signal corresponding to the colors cyan CFP emerald green EmGFP and blue BFP The FP sequences have been cloned into the bacterial expression vector pRSET A to produce pRSET CFP pRSET EmGFP and pRSET BFP For a description of the major features of the vectors see page 2 The pRSET Fluorescent Vectors may be used as follows Remove the Fluorescent Protein gene from the vector by restriction digest for cloning into a mammalian expression vector of choice to create a reporter vector OR Express the Fluorescent Protein in E coli and detect and purify the protein for further study Continued on next page Product Overview Continued Features of PRSET Fluorescent Vectors Green Fluorescent Protein Features of the PRSET Fluorescent Vectors include Bacteriophage T7 promoter for high level inducible expression of the Fluorescent Protein FP in E coli Ribosome binding site RBS optimally spaced from the initiation ATG for effici
5. ent translation of the FP N terminal fusion peptide encoding 6xHis tag for protein purification using metal binding resins Xpress epitope for detection of the expressed fusion protein using an Anti Xpress antibody Enterokinase EK recognition site for efficient cleavage of the fusion peptide from the FP Fluorescent Protein derived from eGFP CFP EmGFP BFP Ampicillin resistance gene for selection in E coli pUC origin for high copy replication and maintenance of the plasmid in E coli Green Fluorescent Protein GFP is a chemi luminescent protein originally isolated from the Aequorea victoria jellyfish Shimomura et al 1962 GFP is a useful biotechnology tool because the gene encoding GFP contains all necessary information for the posttranslational synthesis of the chromophore GFP is widely used as a reporter either when fused to a gene of interest or co expressed in mammalian cells The GFP fluorescence signal is easily detected using fluorescence microscopy and standard filter sets Modifications have been made to wild type GFP to enhance its expression in mammalian systems These modifications include amino acid substitutions that e Change the spectral properties of the protein e Optimize the codon usage for expression in mammalian cells i e enhanced GFP eGFP Zhang et al 1996 Continued on next page Product Overview Continued Modified Fluorescent Proteins FPs that emit fluorescence Fluorescent s
6. es Comm 227 707 711 2009 2010 Life Technologies Corporation All rights reserved 17 Notes invitrogen Corporate Headquarters 5791 Van Allen Carlsb T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
7. f choice We recommend using the PureLink HiPure Plasmid Purification Kit see page 11 Each pRSET FP Vector contains unique restriction sites flanking the FP gene to allow transfer of the FP gene to any vector of choice e g mammalian expression vector using restriction enzyme cloning Refer to the vector map on page 8 to develop your cloning scheme Note If you clone the FP gene into a mammalian vector remember to include a Kozak consensus sequence for proper translation initiation If you are creating a fusion vector remember to clone the FP gene in frame with the gene of interest Continued on next page Expression of Fluorescent Protein in E coli Introduction Important E coli Host for Protein Expression Induction of Protein Expression The pRSET Fluorescent Vectors allow expression of the Fluorescent Protein gene in E coli under the control of the strong bacteriophage T7 promoter The following section provides guidelines for choosing an appropriate E coli strain for transformation of the pRSET Fluorescent Vector and for induction of Fluorescent Protein expression We recommend that you maintain and propagate the plasmid in a recA endA strain of E coli such as TOP10 or DH5a Do not propagate your vector in a BL21 strain of F coli In bacteriophage T7 the T7 promoter drives the expression of gene 10 T7 RNA polymerase recognizes this promoter To express the Fluorescent Protein gene in E coli y
8. ignals at various wavelengths have been created by Proteins introducing amino acid substitutions in the eGFP protein These mutations shift the spectral properties of the protein resulting in cyan CFP emerald EmGFP or blue BFP detected fluorescence Mutations in the FP genes of the pRSET Fluorescent Vectors have been described in a published review Tsien 1998 and are summarized in the table below These mutations are represented by the single letter amino acid abbreviation corresponding to the codon number in the consensus sequence of eGFP followed by the single letter amino acid abbreviation for the substituted amino acid Vector eGFP Mutations pRSET CFP K26R Y66W N146I M153T V163A N164H pRSET EmGFP F64L S65T S72A N149K M153T I167T pRSET BFP F64L Y66H Y145F V163A N198S Mutations listed are as described in the literature When examining the actual sequence the vector codon numbering starts at the first amino acid after the initiation methionine of the FP so that mutations appear to be increased by one position For example the F64L mutation actually occurs in codon 65 of the protein Continued on next page Product Overview Continued Fluorescent Protein Spectral Properties Fluorescent Proteins can be detected using fluorescence microscopy or other methods that use light excitation and detection of emission The table below lists the published excitation and emission waveleng
9. invitrogen pRSET CFP pRSET EmGFP pRSET BFP Vectors Catalog nos V352 20 V353 20 V354 20 Rev date 14 December 2010 Manual part no 25 0842 MAN0000519 Table of Contents Kit Contents and Storage teen tenete tenete rant iv Introduction an een 1 Product Overview e tn sn e HIR RE He iE e sete itn 1 Mello ota 5 General Information sese sneen sener retener nennen 5 Expression of Fluorescent Protein in E coli 6 Purification and Detection of Fluorescent Protein 7 Appendix 25255 on ies ahd re 8 pRSET CFP EmGFP and BFP Vectors een 8 Recipes initial 10 Accessory Products stees eno e tnter 11 Technical Supporte sien rl annan a a a 12 Purchaser Notification essent eene 14 References sit seen deese n Res eR ER See PRO ERR ESL EEEa EEs 17 iii Kit Contents and Storage Shipping and Storage Kit Contents store vectors at 20 C vectors at 20 C pRSET vectors are shipped on wet ice Upon receipt All vectors are supplied as detailed below Store the Catalog Vector Composition Amount no 20 uL of 0 5 ug uL vector in V352 20 pRSET CFP 10 mM Tris HCl 1 mM EDTA 10 pg pH 8 0 20 uL of 0 5 ug uL vector in V353 20 pRSET EmGFP 10 mM Tris HCl 1 mM EDTA 10 ug pH 8 0 20 uL of 0 5 ug uL vector in V354 20 pRSET BFP 10 mM Tris HCl 1 mM EDTA 10 ug pH 8 0 Intended Use For research use only Not intend
10. itzerland and is provided only for use in research In formation about licenses for commercial use is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany This product and its use is the subject of one or more of U S Patent Nos 5 491 084 and 6 146 826 and foreign equivalents This product is sold under license from Columbia University Rights to use this product are limited to research use only and expressly exclude the right to manufacture use sell or lease this product for use for measuring the level of toxicity for chemical agents and environmental samples in cells and transgenic animals No other rights are conveyed Not for human use or use in diagnostic or therapeutic procedures Inquiry into the availability of a license to broader rights or the use of this product for commercial purposes should be directed to Columbia Innovation Enterprise Columbia University Engineering Terrace Suite 363 New York New York 10027 Continued on next page Purchaser Notification Continued Limited Use Label License No 198 Fluorescent Proteins and Stable Cell Lines Expressing Such Pro teins but not for vectors that contain the genes for such fluorescent proteins This product and its use is the subject of one or more of U S Patent Nos 5 777 079 6 066 476 and 6 319 669 and foreign equivalents The purchase of this product conveys to the buyer the nontransferable right to use the purchased amount of the
11. ng clinical trials but excluding without limitation agrochemistry diagnostics environmental veterinary and consumer product applications such as flavors fragrances and taste enhancers The purchase of this product conveys to the purchaser the lim ited non transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser No right to resell this product or any of its compon ents is conveyed expressly by implication or by estoppel This product is for internal research purposes only and is not for use in commercial applications of any kind including without lim itation quality control and commercial services such as report ing the results of purchaser s activities for a fee or other form of consideration For information on obtaining additional rights please contact outlicensing lifetech com or Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 References Shimomura O Johnson F H and Saiga Y 1962 Extraction Purification and Properties of Aequorin a Bioluminescent Protein from the Luminous hHydromedusan Aequorea Journal of Cellular and Comparative Physiology 59 223 239 Tsien R Y 1998 The Green Fluorescent Protein Annu Rev Biochem 67 509 544 Zhang G Gurtu V and Kain S 1996 An Enhanced Green Fluorescent Protein Allows Sensitive Detection of Gene Transfer in Mammalian Cells Biochem Biophys R
12. nt transcription termination fl origin Allows single strand rescue of DNA bla promoter Allows expression of the ampicillin resistance gene Ampicillin ORF Allows selection of the plasmid in E coli pUC origin Allows high copy replication and growth in E coli Recipes LB Medium and Plates Glycerol Stocks 10 LB medium 1 Liter 1 4 Dissolve 10 g tryptone 5 g yeast extract and 10 g NaCl in 950 mL deionized water Adjust the pH of the solution to 7 0 with NaOH and bring the volume up to 1 liter Autoclave on liquid cycle for 20 minutes Allow solution to cool to 55 C and add antibiotic if needed Store at room temperature or at 4 C LB agar plates 1 Prepare LB medium as described above but add 15 g L agar before autoclaving Autoclave on liquid cycle for 20 minutes Allow agar to cool to 55 C and add antibiotic Pour 20 30 mL agar into each 10 cm plate Let agar harden then invert and store at 4 C in the dark Streak a colony out for single colony isolation on LB plates containing the appropriate antibiotic Isolate a single colony and inoculate into 1 2 mL of LB containing the appropriate antibiotic Grow until culture reaches stationary phase Mix 0 85 mL of culture with 0 15 mL of sterile glycerol and transfer to a cryovial Store at 80 C Accessory Products Introduction The following products may be used with the PRSET vectors F
13. or details visit www invitrogen com or contact Technical Support see page 12 Item Amount Catalog no 6 x 2mL precharged prepacked ProBond Im EE resin columns and ProBond Purification System buffers for native and K850 01 denaturing purification ProBond Resin 50 mL R801 01 25 150 mL R801 15 Electrocomp TOP10F 6 x 20 rxns C665 24 ES One Shot TOPIOF Chemically 20 x 50 uL C3030 03 Competent E coli BL21 Star DE3 20 rxns C6010 03 PureLink HiPure Plasmid Miniprep Kit 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 EnterokinaseMax 250 units E180 01 Anti Xpress Antibody 50 uL R910 25 Anti GFP rabbit polyclonal 100 uL A11122 Anti GFP rabbit IgG conjugated to Alexa 100 uL A21311 Fluor 488 Anti GFP mouse monoclonal IgC 100 ug A11120 11 Technical Support Web Resources for Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 USA Tel 1 760 603 7200 Tel Toll Free 1 800 955 6288 Fax 1 760 602 6500 E mail tech_support invitrogen Japanese Headquarters LOOP X Bldg 6F 3 9 15 Kaigan Minato ku Tokyo 108 0022 Tel 81 3 5730 6509 Fax 81 3 5730 6519 E mail jpinfo invitrogen com Visit the Invitrogen website at www invitrogen com Technical resources including manuals vector maps and sequences application notes MSDSs FAQs formulations citations handbooks etc Com
14. ou may use a bacterial host that expresses T7 RNA polymerase or infect the cell with phage expressing 17 RNA polymerase We suggest using a BL21 derived E coli strain as the host for the expression construct These strains express T7 RNA polymerase in a regulated manner For isopropyl D thiogalactoside IPTG induction of protein expression use a BL21 derived strain that contains the DE3 bacteriophage A lysogen The DE3 lysogen contains the T7 RNA polymerase under the control of the lacLIV5 promoter allowing expression of 17 RNA polymerase to be induced by IPTG TM We recommend using BL21 Star DE3 available from Invitrogen see page 11 This strain contains the bacteriophage AX DE3 lysogen Refer to the user manual for the strain you are using for detailed instructions on expressing protein Purification and Detection of Fluorescent Protein Introduction Detection Methods Purification Guidelines Cleavage of the Fusion Peptide by Enterokinase Once you have expressed the FP fusion protein you may verify expression by simply holding the bacterial cell lysate under a UV light source to detect the fluorescent signal Alternatively you may perform western blot analysis to detect the fusion protein using the antibodies listed below Guidelines for protein purification can also be found below You may detect expression of your recombinant fusion protein by western blot using anti Xpress and anti GFP antibodies see
15. page 11 The presence of the polyhistidine 6xHis tag in the fusion peptide of the Fluorescent Protein allows the use of a metal chelating resin such as ProBond or Ni NTA to purify your fusion protein ProBond and Ni NTA are available from Invitrogen see page 11 for ordering Refer to the manual included with each product for instructions to purify your 6xHis tagged fusion protein Note Other metal chelating resins and purification methods are suitable The pRSET Fluorescent Vectors contain an Enterokinase EK recognition site to allow removal of the fusion tag from the expressed FP We recommend using EnterokinaseMax from Invitrogen see page 11 for ordering information Appendix PRSET CFP EmGFP and BFP Vectors Map of pRSET Vectors The map below shows the features of pRSET CFP pRSET EmGFP and pRSET BFP Note that the vectors are identical in size and only differ from one another in the sequence of the Fluorescent Protein gene The vector sequence is available for downloading at www invitrogen com or by contacting Technical Support page 12 BamH EcoRI Nco i aa is Xpress Epitope EME pRSET CFP EmGFP BFP Comments for pRSET CFP EmGFP and BFP 3600 nucleotides T7 promoter priming site bases 9 28 6xHis tag bases 101 118 T7 gene 10 leader bases 122 154 Xpress epitope bases 158 181 EK cleavage site bases 167 181 Fluorescent Protein CFP EmGFP BFP bases 209 928 T7 reve
16. plete technical support contact information Access to the Invitrogen Online Catalog Additional product information and special offers European Headquarters Inchinnan Business Park 3 Fountain Drive Paisley PA4 9RF UK Tel 44 0 141 814 6100 Tech Fax 44 0 141 814 6117 E mail eurotech invitrogen com com Certificate of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box SDS Safety Data Sheets SDSs are available on our website at www invitrogen com sds 12 Continued on next page Technical Support Continued Limited Warranty Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s liability to only
17. rse priming site bases 987 1006 T7 transcription terminator bases 948 1084 f1 origin bases 1148 1603 bla promoter bases 1635 1739 Ampicillin bla resistance gene bases 1734 2594 pUC origin bases 2739 3412 Continued on next page PRSET CFP EmGFP and BFP Vectors Continued Features The pRSET CFP pRSET EmGFP and pRSET BFP vectors 3654 bp contain the following elements Elements have been functionally tested Feature Function T7 promoter Provides tight dose dependent regulation of heterologous gene expression T7 forward Allows sequencing of the insert priming site Ribosome binding Optimally spaced from the cloning region for site RBS efficient translation of the gene of interest Initiation ATG Provides a translational initiation site for the fusion protein 6XHis Tag Permits purification of recombinant fusion protein on metal chelating resins such as ProBond and Ni NTA T7 gene 10 leader Provides protein stability Xpress epitope Allows detection of the fusion protein using an anti Xpress antibody Enterokinase EK recognition site Provides a site for efficient removal of the fusion tag using EKMax Fluorescent Modified fluorescent proteins derived from eGFP Protein CFP whose expression results in the emission of EmGFP or BFP fluorescent signal T7 reverse Allows sequencing of the insert priming site T7 terminator Permits efficie
18. ths for CFP EmGFP and BFP Tsien 1998 All three FPs can be detected with standard FITC filter sets However for optimal detection of the fluorescence signal you may want to use a filter set optimized for detection within the excitation and emission ranges for each FP These filter sets are listed in the table below Vector Excitation Filter Set for Emission Fluorescence nm Microscopy pRSET CFP 452 505 Omega XF114 Chroma 31044 pRSET EmGFP 487 509 Omega XF100 pRSET BFP 308 383 Omega XF10 440 447 Chroma 31021 For information on obtaining filter sets contact Omega Optical Inc www omegafilters com or Chroma Technology Corporation www chroma com Methods General Information Applications E coli Host for Vector Propagation Plasmid Purification Transferring the FP Gene to Another Vector You may use the pRSET Fluorescent Vectors for the following applications e Transfer the Fluorescent Protein gene into a mammalian vector of choice using restriction enzyme cloning See below for guidelines e Express the Fluorescent Protein in E coli and purify the protein See page 7 for guidelines To propagate and maintain pRSET Fluorescent Vectors we recommend using a recA endA strain such as One Shot TOP10F see page 11 for ordering or DH5a Select plasmid containing transformants on LB plates containing 50 100 pg mL ampicillin You may prepare plasmid DNA using your method o
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