pEF-DEST51 - Thermo Fisher Scientific
Contents
1. 5 end of Exon 2 Map and Features of pEF DEST51 Map of The map below shows the elements of pEF DEST51 DNA pEF DEST51 from the entry clone replaces the region between bases 1727 and 3410 The complete sequence of pEF DEST51 is available from our Web site www invitrogen com or by contacting Technical Service page 18 Lng Features of pEF DEST51 7464 nucleotides EF 1a promoter bases 470 1653 T7 promoter bases 1670 1689 attR1 recombination site bases 1720 1844 Chloramphenicol resistance gene bases 1953 2612 ccdB gene bases 2954 3259 attR2 recombination site bases 3300 3424 V5 epitope bases 3450 3491 6xHis tag bases 3501 3518 BGH polyadenylation region 3544 3771 f1 origin bases 3817 4245 SV40 early promoter and origin bases 4250 4594 EM7 promoter bases 4629 4684 Blasticidin resistance gene bases 4703 5101 SV40 early polyadenylation region bases 5259 5389 pUC origin bases 5772 6445 Ampicillin resistance gene b a bases 6590 7450 c bla promoter bases 7451 85 c c complementary strand 15 Map and Features continued Features of pEF DEST51 pEF DEST51 7464 bp contains the following elements All features have been functionally tested Feature Benefit Human elongation factor 1a hEF 1o promoter Allows expression of recombinant proteins in a broad range of mammalian cell types Goldman et al 1996 Mizushima and Nagata 1990 17 promoter All2. For more information refer to our Web site www invitrogen com or contact Technical Service page 18 If you use the pEF GW 51 lacZ plasmid as a positive control vector you may assay for B galactosidase expression by Western blot analysis or activity assay using cell lysates Miller 1972 Invitrogen offers B Gal Antiserum the B Gal Assay Kit and the B Gal Staining Kit for fast and easy detection of B galactosidase expression see page v for ordering information The C terminal peptide containing the V5 epitope and the polyhistidine region will add approximately 5 kDa to your protein The presence of the C terminal polyhistidine 6xHis tag in your recombinant fusion protein allows use of a metal chelating resin such as ProBond to purify your fusion protein The ProBond Purification System and bulk ProBond resin are available from Invitrogen see page vi for ordering information Refer to the ProBond Purification System manual for protocols to purify your fusion protein Invitrogen also offers Ni NTA Agarose Catalog no R901 01 for purification of proteins containing a polyhistidine 6xHis tag Note Other metal chelating resins and purification methods are suitable Creating Stable Cell Lines Introduction 4 SECO 2 Z g Nou N Blasticidin Blasticidin Selection Guidelines 10 The pEF DEST51 vector contains the blasticidin resistance gene to allow selection of stable
3. Technical resources including manuals vector maps and sequences application notes SDSs FAQs formulations citations handbooks etc e Complete technical support contact information e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail jpinfo invitrogen com E mail tech_support invitrogen com eurotech invitrogen com SDS Certificate of Analysis 18 Safety Data Sheets SDSs are available on our website at www invitrogen com sds 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 continued on next page Technical Service co
4. CACAGGTAAG TGCCGTGTGT GCGTGCCTTG AATTACTTCC TTGGAAGTGG GTGGGAGAGT GAGTTGAGGC CTGGCCTGGG CCTGTCTCGC TGCTTTCGAT CGCTTTTTTT CTGGCAAGAT GTCGGCAATT GTGTACTGGC GCCGTGAACG GGTTCCCGCG ACCTGGCTGC TCGAGGCCTT CGCTGGGGCC AAGTCTCTAG AGTCTTGTAA Sp1 CGGTTTTTGG Spi GGceeeqdccT GCGAGCGCGG CTGCTCTGGT GCCTGGCCTC CCCGGTCGGC ACCAGTTGCG GCTCAAAATG GAGGACGCGG AAAGGGCCTT TCCGTCCTCA CCAGGCACCT CGATTAGTTC GGTTTTATGC GATGGAGTTT GGCACTTGAT GTAATTCTCC TCAAGCCTCA GACAGTGGTT GGCCQCGGGC GGCGA CGGGG CCACCGAGAA GCGCCGCCGT TGAGCGGAAA CGCTCGGGAG GCCGTCGCTT TCGAGCTTTT CCCCACACTG TTGGAATTTG CAAAGTTTTT GAACCGGTGC CTAGAGAAGG TCCGCCTTTT TCCCGAGGGT Start of Transcription TTCTTTTTCG CAACGGGTTT CCCACAGTCC TGGCGCGGGG GGGGGAGAAC GCCGCCAGAA GGCCTGGCCT CTTTACGGGT AGTACGTGAT TCTTGATCCC GCGCTTAAGG AGCCCCTTCG GCCGCGTGCG AATCTGGTGG CCATTTAAAA TTTTTGATGA ATGCGGGCCA AGATCTGCAC CCCGTGCGTC CCAGCGCACA TCGGACGGGG GTAGTCTCAA Sp 1 Exon TATGGCCCTT GAGCTTCGGG CCTCGTGCTT CACCTTCGCG CCTGCTGCGA ACTGGTATTT TGTTCGGdGA GCTGGCCGGC Sp 1 GTATcGcccc ccccreeeco GCAABGCTGG GATGGCCGCT TCCCGGCCCT Sp 1 AGCGGGCGGG TGAGTCACCC Ap 1 CATG IGACTC CACGGAGTAC GGAGTACGTC GTCTTTAGGT AGTGGGTGGA GACTGAAGTT CCCTTTTTGA GTTTGGATCT 3 end of Intron 1 TTCTTCCATT TCAGGTGTCG GCTGCAGGGA ACACAAAGGA CGGGCGCCGT TGGGGGGAGG AGGCCAGCTT TGGTTCATTC TGA
5. T and Hanaoka F 1991 Blasticidin S Resistance Gene bsr A Novel Selectable Marker for Mammalian Cells Exp Cell Res 197 229 233 Kimura M Takatsuki A and Yamaguchi I 1994 Blasticidin S Deaminase Gene from Aspergillus terreus BSD A New Drug Resistance Gene for Transfection of Mammalian Cells Biochim Biophys ACTA 1219 653 659 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biology 115 887 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 Landy A 1989 Dynamic Structural and Regulatory Aspects of Lambda Site specific Recombination Annu Rev Biochem 58 913 949 continued on next page 22 References continued Lindner P Bauer K Krebber A Nieba L Kremmer E Krebber C Honegger A Klinger B Mocikat R and Pluckthun A 1997 Specific Detection of His tagged Proteins With Recombinant Anti His Tag scFv Phosphatase or scFv Phage Fusions BioTechniques 22 140 149 Miller J H 1972 Experiments in Molecular Genetics Cold Spring Harbor New York Cold Spring Harbor Laboratory Mizushima S and Nagata S 1990 pEF BOS a Powerful Mammalian Expression Vector Nuc
6. cell lines To create stable cell lines transfect your construct into the mammalian cell line of choice and select for foci using blasticidin General guidelines are provided below To obtain stable transfectants we recommend that you linearize your pEF DEST51 construct before transfection While linearizing the vector may not improve the efficiency of transfection it increases the chances that the vector does not integrate in a way that disrupts elements necessary for expression in mammalian cells To linearize your construct cut at a unique site that is neither located within a critical element nor within your gene of interest Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells Takeuchi et al 1958 Yamaguchi et al 1965 Resistance is conferred by expression of either one of two blasticidin S deaminase genes bsd from Aspergillus terreus Kimura et al 1994 or bsr from Bacillus cereus Izumi et al 1991 These deaminases convert blasticidin S to a non toxic deaminohydroxy derivative Izumi et al 1991 Blasticidin is available separately from Invitrogen see page v for ordering information Use as follows 1 Prepare blasticidin in sterile water and filter sterilize the solution 2 Use 2 5 to 10 pg ml of blasticidin in complete medium 3 Test varying concentrations of blasticidin on your cell line t
7. for protein concentration Note Do not use protein assays utilizing Coomassie Blue or other dyes NP 40 interferes with the binding of the dye with the protein 6 Add SDS PAGE sample buffer see page 12 for a recipe to a final concentration of 1X and boil the sample for 5 minutes 7 Load 20 pg of lysate onto an SDS PAGE gel and electrophorese Use the appropriate percentage of acrylamide to resolve your fusion protein To facilitate separation and visualization of your recombinant fusion protein by polyacrylamide gel electrophoresis a wide range of pre cast NuPAGE and Novex Tris Glycine polyacrylamide gels and electrophoresis apparatus are available from Invitrogen For more information refer to our Web site www invitrogen com or contact Technical Service page 18 continued on next page Expression and Analysis continued Detecting Recombinant Fusion Proteins Assay for p galactosidase Note Purification of Recombinant Fusion Proteins To detect expression of your recombinant fusion protein by Western blot analysis you may use the Anti V5 antibodies or the Anti His C term antibodies available from Invitrogen see page v for ordering information or an antibody to your protein of interest In addition the Positope Control Protein Catalog no R900 50 is available from Invitrogen for use as a positive control for detection of fusion proteins containing a V5 epitope or a polyhistidine 6xHis tag
8. respective owners 23 Notes invitrogen by A technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 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
9. tag requires Anti His C term HRP the free carboxyl group for R931 25 Antibody detection Lindner et al 1997 HHHHHH COOH Anti His C term AP R932 25 Antibody continued on next page Accessory Products continued Purification of If your gene of interest is in frame with the C terminal Recombinant peptide containing the V5 epitope and the polyhistidine Fusion Protein 6xHis tag you may use Immobilized Metal Affinity Chromatography IMAC to purify your recombinant fusion protein The ProBond Purification System or bulk ProBond resin are available separately from Invitrogen See the table below for ordering information Product Quantity Catalog no ProBond Nickel chelating Resin 50 ml R801 01 150 ml R801 15 ProBond Purification System 6 purifications K850 01 ProBond Purification System with 1 kit K853 01 Anti His C term HRP Antibody ProBond Purification System with 1 kit K854 01 Anti V5 HRP Antibody Purification Columns 50 R640 50 10 ml polypropylene columns vi Overview Description Features Methods pEF DEST51 is a 7 5 kb vector derived from pEF6 V5 His and adapted for use with the Gateway Technology It is designed to allow high level constitutive expression in a variety of mammalian hosts pEF DEST51 contains the following elements Human elongation factor 1a subunit promoter hEF 1o for high le
10. 008 Phone 760 603 7200 or e mail EF lalpha promoter products are sold under license for research purposes only The use of this product for any commercial purpose including but not limited to use in any study for the purpose of a filing of a new drug application requires a license from Mochida Pharmaceutical Co Ltd 7 Yotsuya 1 Chome Shinjuku Ku Tokyo 160 Japan Tel 81 3 3225 5451 Fax 81 3 3225 6091 21 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology New York Greene Publishing Associates and Wiley Interscience Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Molec Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nucleic Acids Res 15 1311 1326 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 115 121 Felgner P L a and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 Goodwin E C and Rottman F M 1992 The 3 Flanking Sequence of the Bovine Growth Hormone Gene Contains Novel Elements Required for Efficient and Accurate Polyadenylation J Biol Chem 267 16330 16334 Izumi M Miyazawa H Kamakura T Yamaguchi I Endo
11. 03 7200 continued on next page Purchaser Notification continued Limited Use Label License No 51 Blasticidin and the Blasticidin Selection Marker Limited Use Label License No 54 ULB ccdB Selection Technology Limited Use Label License No 60 EF 1o Promoter Blasticidin and the blasticidin resistance gene bsd are the subject of U S Patent No 5 527 701 sold under patent license for research purposes only For information on purchasing a license to this product for purposes other than research contact Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 Fax 760 602 6500 This product is the subject of one or more of U S Patent Numbers 5 910 438 6 180 407 and 7 176 029 and corresponding foreign patents and is sold under license from the Universit Libre de Bruxelles for research purposes only ccdB selection technology is described in Bernard et al Positive Selection Vectors Using the F Plasmid ccdB Killer Gene Gene 148 1994 71 74 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity For licensing information for use in other than research please contact Out Licensing Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92
12. 10 200 ug DNA Catalog no K1910 01 or CsCl gradient centrifugation For established cell lines e g HeLa consult original references or the supplier of your cell line for the optimal method of transfection We recommend that you follow exactly the protocol for your cell line Pay particular attention to medium requirements when to pass the cells and at what dilution to split the cells Further information is provided in Current Protocols in Molecular Biology Ausubel et al 1994 Methods for transfection include calcium phosphate Chen and Okayama 1987 Wigler et al 1977 lipid mediated Felgner et al 1989 Felgner and Ringold 1989 and electroporation Chu et al 1987 Shigekawa and Dower 1988 If you wish to use a cationic lipid based reagent for transfection we recommend using Lipofectamine 2000 Reagent available from Invitrogen see page v for ordering information For more information refer to our Web site www invitrogen com or contact Technical Service page 18 continued on next page Transfection continued Positive Control pEF GW 51 lacZ is provided as a positive control vector for mammalian cell transfection and expression see page 17 for a map and may be used to optimize recombinant protein expression levels in your cell line The vector allows expression of a C terminally tagged B galactosidase fusion protein that may be detected by Western blot or functional assay To propagate and
13. F DEST51 Vector lyophilized in TE 6 ug pH 8 0 pEF GW 51 lacZ lyophilized in TE 10 ug Control Plasmid pH 8 0 Accessory Products Additional Additional products that may be used with the pEF DEST51 Products Gateway Vector are available from Invitrogen Ordering information is provided below Product Amount Catalog no Gateway LR Clonase Enzyme Mix 20 reactions 11791 019 One Shot TOP10 Chemically 10 reactions 4040 10 KEBIpetent cens 20 reactions C4040 03 One Shot TOP10 Electrocompetent 10 reactions C4040 50 Cells 20 reactions C4040 52 Lipofectamine 2000 Reagent 15ml 11668 019 0 75 ml 11668 027 Blasticidin 50 mg R210 01 p Gal Antiserum 50 ul R901 25 B Gal Assay Kit 100 reactions K1455 01 B Gal Staining Kit 1 kit K1465 01 Amount supplied is sufficient for 25 Westerns using 10 ml working solution per reaction Detection of Expression of your recombinant fusion protein can be Recombinant detected using an antibody to the appropriate epitope The Proteins amount of antibody supplied is sufficient for 25 Westerns Product Epitope Catalog no Anti V5 Antibody Detects 14 amino acid epitope R960 25 Anti V5 HRP Antibody derived from the P and V proteins R961 25 of the paramyxovirus SV5 Anti V5 AP Antibody Southern et al 1991 R962 25 GKPIPNPLLGLDST Anti His C term Detects the C terminal R930 25 Antibody polyhistidine 6xHis
14. 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 diagnostic 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 Technologies Corporation will not assert a claim against the buyer of infringement of the above patents based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research 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 used in the manufacture of such product Life Technologies Corporation will not assert a claim against the buyer of infringement of the above patents based upon the use of this product to manufacture a protein for sale provided that no method claim in the above patents was used in the manufacture of such protein If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies 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 Licensing Department Life Technologies Corporation 5791 Van Allen Way Carlsbad California 92008 Phone 760 6
15. e use of Clonase purchased from Life Technologies Corporation or its authorized distributors The buyer cannot modify the recombination sequence s contained in this product for any purpose 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 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 license from Life Technologies 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 Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its
16. in a hood Preparing and Blasticidin may be obtained separately from Invitrogen Storing Stock Catalog no R210 01 in 50 mg aliquots Blasticidin is soluble Solutions in water Sterile water is generally used to prepare stock solutions of 5 to 10 mg ml Dissolve blasticidin in sterile water and filter sterilize the solution Aliquot in small volumes suitable for one time use and freeze at 20 C for long term storage or store at 4 C for short term storage Aqueous stock solutions are stable for 1 2 weeks at 4 C and 6 8 weeks at 20 C pHofthe aqueous solution should be 7 0 to prevent inactivation of blasticidin Do not subject stock solutions to freeze thaw cycles do not store in a frost free freezer Upon thawing use what you need and store the thawed stock solution at 4 C for up to 2 weeks Medium containing blasticidin may be stored at 4 C for up to 2 weeks 13 Human EF 1o Promoter Description 14 461 521 581 641 701 761 821 881 941 1001 1061 1121 1181 1241 1301 1361 1421 1481 1541 1601 The diagram below shows all the features of the EF 1a promoter used in Invitrogen pEF vectors Mizushima and Nagata 1990 Uetsuki et al 1989 m 5 end of human EF 1a promoter GGAGTGCCTC GTGAGGCTCC GGTGCCCGTC AGTGGGCAGA GCGCACATCG CCGAGAAGTT GGGGGGAGGG TAAACTGGGA AAGTGATGTC TATA box CGTATATAAG TGCAGTAGTC 5 end of Intron 1
17. into multiple vector systems To express your gene of interest using Gateway cloning technology simply 1 Clone your gene of interest into a Gateway entry vector to create an entry clone 2 Generate an expression clone by performing an LR recombination reaction between the entry clone and a Gateway destination vector e g pEF DEST51 3 Transfect your expression clone into the cell line of choice for transient or constitutive expression of your gene of interest For more information on the Gateway System refer to the Gateway Technology Manual This manual is available for downloading from our Web site www invitrogen com or by contacting Technical Service page 18 Using pEF DEST51 Important Propagating pEF DEST51 Resuspending pEF DEST51 Entry Clone The pEF DEST51 vector is supplied as a supercoiled plasmid Although Invitrogen has previously recommended using a linearized destination vector for more efficient recombination further testing has found that linearization of this vector is NOT required to obtain optimal results for any downstream application If you wish to propagate and maintain pEF DEST51 we recommend using Library Efficiency DB3 1 Competent Cells Catalog no 11782 018 from Invitrogen for transformation The DB3 1 E coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene Note DO NOT use general E coli clo
18. invitrogen by technologies pEF DEST51 Gateway Vector A destination vector for cloning and expression of C terminal fusion proteins in mammalian cells Catalog no 12285 011 Rev Date 7 July 2010 Manual part no 25 0428 MAN0000240 Table of Contents Important Information sseeeeee eee nnne nenne iv Accessory Products jn M 1 OVervie Ws Lir ie tette vtt d eet eto M Lb t Lc 1 Using pBE DEST 51 etn eec enger 3 Transfection soe ose e SERIEN STi HR EPIRI ede eR en HERES ar i 6 Expression and Analysis sees 8 Creating Stable Cell Lines riter tenete intet 10 f p qe 11 Rectpesui ss RH EUR UE RIEN ERAI S 11 Blasticidin iso rrr E RERO RENE pe EORR M HERES E UHR 13 Human BE Id Promoter suus deg uuu 14 Map and Features of pEF DEST51 sss 15 Map Of pEF GW 51 71aCZ iie o itte reip ro o dro ce eene 17 TTechinical Service rne deeem pte Rt repe En etes 18 Purchaser Notification dte reo dete ee eei eie e e IR 20 Referefhces etate etatem o a den ba e T eb teta 22 Important Information Shipping and pEF DEST51 and pEF GW 51 lacZ are shipped at room Storage temperature Upon receipt store at 20 C Products are guaranteed for six months from date of shipment when stored properly Contents The pEF DEST51 Gateway Vector components are listed below Item Concentration Volume pE
19. leic Acids Res 18 5322 Shigekawa K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells BioTechniques 6 742 751 Southern J A Young D F Heaney F Baumgartner W and Randall R E 1991 Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics J Gen Virol 72 1551 1557 Takeuchi S Hirayama K Ueda K Sakai H and Yonehara H 1958 Blasticidin S A New Antibiotic The Journal of Antibiotics Series A 11 1 5 Uetsuki T Naito A Nagata S and Kaziro Y 1989 Isolation and Characterization of the Human Chromosomal Gene for Polypeptide Chain Elongation Factor 1a J Biol Chem 264 5791 5798 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 Yamaguchi H Yamamoto C and Tanaka N 1965 Inhibition of Protein Synthesis by Blasticidin S I Studies with Cell free Systems from Bacterial and Mammalian Cells J Biochem Tokyo 57 667 677 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their
20. mM and the pH must be 7 0 continued on next page Using pEF DEST51 continued Confirming the Expression Clone Recombination Region 1641 1701 3402 3450 3498 The ccdB gene mutates at a very low frequency resulting in a very low number of false positives True expression clones will be ampicillin resistant and chloramphenicol sensitive Transformants containing a plasmid with a mutated ccdB gene will be both ampicillin and chloramphenicol resistant To check your putative expression clone test for growth on LB plates containing 30 pg ml chloramphenicol A true expression clone will not grow in the presence of chloramphenicol The recombination region of the expression clone resulting from pEF DEST51 x entry clone is shown below Features of the Recombination Region e Shaded regions correspond to those DNA sequences transferred from the entry clone into pEF DEST51 by recombination Non shaded regions are derived from the pEF DEST51 vector The nucleotides on either side of the shaded region correspond to bases 1727 and 3410 respectively of the pEF DEST51 vector sequence EF 1a promoter TCAGGTGTCG TGAGGAATTA GCTTGGTACT AATACGACTC ACTATAGGGA GACCCAAGCT AGTCCACAGC ACTCCTTAAT CGAACCATGA TTATGCTGAG TGATATCCCT CTGGGTTCGA 1727 l GGCTAGGTAA GCTTGATCAA CAAGTTTGTA CAAAAAAGCA GGCTN GENE MAE CCGATCCATT CGAACTAGTT GTTCAAACAT GTTTTTTOGT GEGEN Ic MET poji NTG pr
21. maintain the plasmid 1 Resuspend the vector in 10 ul sterile water to prepare a 1 ug l stock solution Use the stock solution to transform a recA endA E coli strain like TOP10 DH5a JM109 or equivalent 2 Select transformants on LB agar plates containing 50 100 ug ml ampicillin 3 Prepare a glycerol stock of a transformant containing plasmid for long term storage Expression and Analysis Introduction Preparation of Cell Lysates Polyacrylamide Gel Electrophoresis Expression of your gene of interest from the expression clone can be performed in either transiently transfected cells or stable cell lines see page 10 for guidelines to create stable cell lines A sample protocol is provided below Other protocols are suitable To lyse cells 1 Wash cell monolayers 5 x 105 to 1 x 106 cells once with phosphate buffered saline PBS available from Gibco Catalog no 10010 023 2 Scrape cells into 1 ml PBS and pellet the cells at 1500 x g for 5 minutes 3 Resuspend in 50 ul Cell Lysis Buffer see page 12 for a recipe Other cell lysis buffers are suitable Vortex 4 Incubate cell suspension at 37 C for 10 minutes to lyse the cells Note You may prefer to lyse the cells at room temperature or on ice if degradation of your protein is a potential problem 5 Centrifuge the cell lysate at 10 000 x g for 10 minutes at 4 C to pellet nuclei and transfer the supernatant to a fresh tube Assay the lysate
22. ning strains including TOP10 or DH5a for propagation and maintenance as these strains are sensitive to CcdB effects Before you perform the LR Clonase reaction resuspend pEF DEST51 to 50 150 ng ul in sterile water To recombine your gene of interest into pEF DEST51 you should have an entry clone containing your gene of interest For your convenience Invitrogen offers the pENTR Directional TOPO Cloning Kit Catalog no K2400 20 for 5 minute cloning of your gene of interest into an entry vector For more information on entry vectors available from Invitrogen refer to our Web site www invitrogen com or contact Technical Service page 18 For detailed information on constructing an entry clone refer to the specific entry vector manual For detailed information on performing the LR recombination reaction refer to the Gateway Technology Manual continued on next page Using pEF DEST51 continued Points to Consider Before Recombining Recombining Your Gene of Interest Note Your insert should contain a Kozak consensus sequence with an ATG initiation codon for proper initiation of translation Kozak 1987 Kozak 1991 Kozak 1990 An example of a Kozak consensus sequence is provided below Other sequences are possible but the G or A at position 3 and the Gat position 4 are the most critical for function shown in bold The ATG initiation codon is shown underlined G AJNNATGG If you wish to incl
23. ntinued 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 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 f
24. o ala COA Ger GGT CGA attB1 3410 phe leu tyr lys val val asp leu glu gly pro arg phe glu TTC TTG TAC AAA GTG GTT GAT CTA GAG GGC CCG CGG TTC GAA AAG AAC ATG TTT CAC CAA CTA GAT CTC CCG GGC GCC AAG CTT attB2 V5 epitope gly lys GGT AAG CCA TTC pro ile pro asn pro leu leu gly leu asp ser thr arg thr CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG CGT ACC GGA TAG GGA TTG GGA GAG GAG CCA GAG CTA AGA TGC GCA TGG 6xHis tag gly his GGT CAT CCA GTA his his his his his CAT CAC CAT CAC CAT TGA GTTTAAACC CGCTGATCAG CCTCGACTGT GTA GTG GTA GTG GTA ACT CAAATTTGG GCGACTAGTC GGAGCTGACT Transfection Introduction Plasmid Preparation Methods of Transfection This section provides general information for transfecting your expression clone into the mammalian cell line of choice We recommend that you include the positive control vector pEF GW 51 lacZ and a mock transfection negative control in your experiments to evaluate your results Once you have generated your expression clone you must isolate plasmid DNA for transfection Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride Contaminants will kill the cells and salt will interfere with lipid complexing decreasing transfection efficiency We recommend isolating plasmid DNA using the S N A P MiniPrep Kit 10 15 ug DNA Catalog no K1900 01 the S N A P MidiPrep Kit
25. o determine the concentration that kills your cells kill curve Cells differ in their susceptibility to blasticidin Complete selection can take up to 10 days of growth in selective medium Refer to the Appendix page 13 for instructions on how to prepare and store blasticidin Recipes LB Luria Bertani Medium and Plates Low Salt LB Medium with Blasticidin Appendix Composition 1 0 Tryptone 0 5 Yeast Extract 1 0 NaCl pH 7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 10 g NaCl in 950 ml deionized water 2 Adjust the pH of the solution to 7 0 with NaOH and bring the volume up to 1 liter 3 Autoclave on liquid cycle for 20 minutes at 15 psi Allow solution to cool to 55 C and add antibiotic 100 ug ml ampicillin if needed 4 Store at room temperature or at 4 C LB agar plates 1 Prepare LB medium as above but add 15 g L agar before autoclaving 2 Autoclave on liquid cycle for 20 minutes at 15 psi 3 After autoclaving cool to 55 C add antibiotic 100 ug ml of ampicillin and pour into 10 cm plates 4 Let harden then invert and store at 4 C Low Salt LB Medium 10 g Tryptone 5 g NaCl 5 g Yeast Extract 1 Combine the dry reagents above and add deionized distilled water to 950 ml Adjust pH to 7 0 with 1 N NaOH Bring the volume up to 1 liter For plates add 15 g L agar before autoclaving 2 Autoclave on liquid cycle at 15 psi and 121 C for 20 minute
26. or 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 Invitrogen assumes 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 19 Purchaser Notification Introduction Limited Use Label License No 19 Gateway Cloning Products 20 Use of the pEF DEST51 Gateway Vector is covered under the licenses detailed below The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The purchase of this product does not convey a license under any method claims in the foregoing patents or patent applications or to use this product with any recombination sites other than those purchased from Life Technologies Corporation or its authorized distributor The right to use methods claimed in the foregoing patents or patent applications with this product for research purposes only can only be acquired by th
27. ows in vitro transcription in the sense orientation attR1 and attR2 sites Allows recombinational cloning of the gene of interest from an entry clone Chloramphenicol resistance gene Allows counterselection of expression clones ccdB gene Allows negative selection of expression clones V5 epitope Allows detection of recombinant fusion proteins by the Anti V5 antibodies Southern et al 1991 C terminal polyhistidine tag Allows purification of recombinant proteins on metal chelating resin such as ProBond Allows detection of the recombinant protein by the Anti His C term antibodies Lindner et al 1997 Bovine growth hormone Allows efficient transcription termination and BGH polyadenylation polyadenylation of mRNA Goodwin and signal Rottman 1992 f1 origin Allows rescue of single stranded DNA SV40 early promoter and Allows high level expression of the blasticidin origin resistance gene and episomal replication in cells expressing the SV40 large T antigen EM7 promoter Allows expression of the blasticidin resistance gene in E coli Blasticidin resistance gene Allows selection of stable transfectants in mammalian cells Kimura et al 1994 SV40 early polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA pUC origin Allows high copy number replication and growth in E coli Ampicillin resistance gene Allo
28. s 3 Allow the medium to cool to at least 55 C before adding the blasticidin to 100 ug ml final concentration 4 Store plates at 4 C in the dark Plates containing blasticidin are stable for up to 2 weeks continued on next page 11 Recipes continued Cell Lysis Buffer 4X SDS PAGE Sample Buffer 12 50 mM Tris pH 7 8 150 mM NaCl 1 Nonidet P 40 1 This solution can be prepared from the following common stock solutions For 100 ml combine 1M Tris base 5 ml 5M NaCl 3 ml Nonidet P 40 1ml 2 Bring the volume up to 90 ml with deionized water and adjust the pH to 7 8 with HCl 3 Bring the volume up to 100 ml Store at room temperature To prevent proteolysis you may add 1 mM PMSF 1 uM leupeptin or 0 1 uM aprotinin before use 1 Combine the following reagents 0 5 M Tris HCl pH 6 8 5 ml Glycerol 100 4 ml B mercaptoethanol 0 8 ml Bromophenol Blue 0 04 g SDS 0 8 g 2 Bring the volume to 10 ml with sterile water 3 Aliquot and freeze at 20 C until needed Blasticidin Molecular The formula for blasticidin S is Ci7H2s5NgOs HCl and the Weight molecular weight is 458 9 The diagram below shows the Formula and structure of blasticidin Structure ae Sn A N HOOC o HC1 CH3 Y 7YY NH NH2 O Handling Always wear gloves mask goggles and protective clothing Blasticidin e g a laboratory coat when handling blasticidin Weigh out blasticidin and prepare solutions
29. ude the V5 epitope and 6xHis tag your gene in the entry clone should not contain a stop codon In addition the gene should be designed to be in frame with the C terminal epitope tag after recombination Refer to the Recombination Region on the next page If you do NOT wish to include the V5 epitope and 6xHis tag your gene should contain a stop codon in the entry clone Each entry clone contains attL sites flanking the gene of interest Genes in an entry clone are transferred to the destination vector backbone by mixing the DNAs with the Gateway LR Clonase enzyme mix see page v for ordering information The resulting recombination reaction is then transformed into E coli and the expression clone selected Recombination between the attR sites on the destination vector and the attL sites on the entry clone replaces the ccdB gene and the chloramphenicol Cm gene with the gene of interest and results in the formation of attB sites in the expression clone Follow the instructions in the Gateway Technology Manual to set up the LR Clonase reaction transform E coli and select for the expression clone The presence of the EM7 promoter and the blasticidin resistance gene allows for selection of E coli transformants using blasticidin For selection use Low Salt LB agar plates containing 100 pg ml blasticidin see page 11 for a recipe For blasticidin to be active the salt concentration of the medium must remain low lt 90
30. vel expression across a broad range of species and cell types see page 14 Two recombination sites attR1 and attR2 downstream of the EF 1a promoter for recombinational cloning of the gene of interest from an entry clone Chloramphenicol resistance gene located between the two attR sites for counterselection The ccdB gene located between the two attR sites for negative selection The V5 epitope and 6xHis tag for detection and purification optional Bovine growth hormone BGH polyadenylation sequence for proper termination and processing of the recombinant transcript f1intergenic region for production of single strand DNA in F plasmid containing E coli SV40 early promoter and origin for expression of the blasticidin resistance gene and stable propagation of the plasmid in hosts expressing the SV40 large T antigen EM7 promoter for expression of the blasticidin resistance gene in E coli Blasticidin resistance gene for selection of stable cell lines The pUC origin for high copy replication and maintenance of the plasmid in E coli The ampicillin bla resistance gene for selection in E coli For a map of pEF DEST51 see page 15 continued on next page Overview continued The Gateway Technology Gateway is a universal cloning technology that takes advantage of the site specific recombination properties of bacteriophage lambda Landy 1989 to provide a rapid and highly efficient way to move your gene of interest
31. ws selection of transformants in E coli 16 Map of pEF GW 51 lacZ Description pEF GW 51 lacZ is an 8904 bp control vector containing the gene for B galactosidase pEF GW 51 lacZ was constructed using the Gateway LR recombination reaction between an entry clone containing the lacZ gene and pEF DEST51 P galactosidase is expressed as a fusion to the C terminal tag The fusion protein is approximately 121 kDa in size Map of The map below shows the elements of pEF GW 51 lacZ pEF GW The complete sequence of pEF GW 51 lacZ is available 51 lacZ from our Web site www invitrogen com or by contacting Technical Service page 18 V5 6xHis Stop Features of pEF GW 51 lacZ 8904 nucleotides EF 1a promoter bases 470 1653 T7 promoter bases 1670 1689 attB1 recombination site bases 1720 1744 lacZ ORF bases 1764 4823 attB2 recombination site bases 4840 4864 V5 epitope bases 4890 4931 6xHis tag bases 4941 4958 BGH polyadenylation region bases 4984 5211 f1 origin bases 5257 5685 SV40 early promoter and origin bases 5690 6034 EM7 promoter bases 6069 6124 Blasticidin resistance gene bases 6143 6541 SV40 early polyadenylation region bases 6699 6829 pUC origin bases 7212 7885 Ampicillin resistance gene b a bases 8030 8890 c bla promoter bases 8891 85 c c 2 complementary strand 17 Technical Service Web Resources Visit the Invitrogen website at www invitrogen com for
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