pEF1/V5-His - Thermo Fisher Scientific
Contents
1. 1 Consult the multiple cloning sites described on pages 3 5 to determine which vector A B or C should be used to clone your gene in frame with the C terminal V5 epitope and polyhistidine tag 2 Ligate your insert into the appropriate vector and transform into E coli Select transformants on 50 to 100 pg mL ampicillin 3 Analyze your transformants for the presence of insert by restriction digestion Select a transformant with the correct restriction pattern and confirm that your gene is in frame with the C terminal peptide by sequencing Transfect your construct into the cell line of choice Test for expression of your recombinant gene by western blot analysis or functional assay If you do not have an antibody to your protein Invitrogen offers the Anti V5 Antibody or the Anti His C term Antibody to detect your recombinant protein See page 14 for more information 7 To purify your recombinant protein you may use metal chelating resin such as ProBond ProBond resin is available separately Methods Cloning into pEF1 V5 His A B and C General Molecular Biology Techniques E coli Strain Maintaining pEF1 V5 His Cloning Considerations For help with DNA ligations E coli transformations restriction enzyme analysis purification of single stranded DNA DNA sequencing and DNA biochemistry refer to Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology2. Ausubel et al 1994 Many E coli strains are suitable for the growth of this vector including TOP10F DH5aF JM109 and INVaF We recommend that you propagate vectors containing inserts in E coli strains that are recombination deficient recA and endonuclease A deficient endA To propagate and maintain the pEF1 V5 His vectors use a small amount of the supplied 0 5 ug uL stock solution in TE pH 8 0 to transform a recA endA E coli strain like TOP10F DH5a JM109 or equivalent Select transformants on LB plates containing 50 100 pg mL ampicillin Be sure to prepare a glycerol stock of each plasmid for long term storage Your insert should contain a Kozak consensus sequence with an ATG initiation codon for proper initiation of translation Kozak 1987 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 G at position 4 shown in bold illustrates the most commonly occurring sequence with strong consensus Replacing one of the two bases at these positions provides moderate consensus while having neither results in weak consensus The ATG initiation codon is shown underlined G A NNATGG If you wish to express your protein WITHOUT the C terminal peptide be sure to include a stop codon Continued on next page Cloning into pEF1 V5 His A B and C Continued Multiple Cloning Site of Version A 1579 1659 1733
3. 1 RE VESEN CGG TTC GAA GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG CGT ACC GGT CAT CAT Arg Phe Glu Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His region Pme BGH Reverse priming site m 1 CAC CAT CAC CAT TGA GTTTAAAC CCGCTGATCA GCCTCGACTG TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT His His His His BGH polyadenylation signal Me we GCCCCTCCCC CGTGCCTTCC TTGACCCTGG AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA TTCTATTCTG GGGGGTGGGG TGGGGCAGGA CAGCAAGGGG GAGGATTGGG AAGACAATAG Continued on next page Cloning into pEF1 V5 His A B and C Continued Multiple Cloning Site of Version C Below is the multiple cloning site for pEF1 V5 His C Restriction sites are labeled to indicate the cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing For more information on the hEF 1a promoter see page 9 3 end of hEF 1a Intron 1 TAA GC TTG G1 Leu Val Pro Ser Ser Asp Pro Leu GCG GCC GCT Ala Ala Ala BGH polyadenylation si rd 1 ACG CGT ACC Thr Arg Thr 1579 GTTTGGATCT TGGTTCATTC TCAAGCCTCA GACAGTGGTT CAAAGTTTTT TTC T7 promoter priming site 1 1659 GCTTGGTACT AATACGACTC ACTATAGGGA GACCCAAGCT GGCTAGGI BstX1 EcoRI EcoRV BstX Not I I j 1735 GTC CAG TGT GGT GGA ATT CTG CAG ATA TCC AGC ACA GTG
4. 1799 1865 1939 2019 GTTTGGATCT TGGTTCATT Below is the multiple cloning site for pEF1 V5 His A Restriction sites are labeled to indicate the cleavage site The boxed nucleotides indicate the variable region Note that there is a stop codon between the Spe I site and the BstX I site The multiple cloning site has been confirmed by sequencing and functional testing For more information on the hEF 1a promoter see page 9 T7 promoter priming site f GCTTGGTACT AATACGACT TAG TCC AGT Ser Ser BstB BstX EcoR I l GTG GTG GAA Val Val Glu re ACT TTC Phe TGC AGA Cys Arg V5 epitope TC TCAAGCCTCA GACAGTGGTT 1 TATAGGGA GACCCAAGCT EcoR V TAT Tyr BstX l CCA GCA CAG TGG CGG Ala Gln Trp Arg Pro GGCTAGGTAA GCT TGG TAC CGA GCT 3 end of hEF 1a Intron 1 5 end of h Kpn Trp Tyr Arg Ala Not I Xba CAAAGTTTTT TICTTCCATT TCAGGTGTCG TGAGGAATTA EF 1a Exon 2 BamH Spe l i CGG ATC CAC Arg Ile His i CCG CTC GAG TCI I AGA GGG CCC r TTC GAA GGT Phe Glu Gly region KE OS SSE CAT CAC CAT His His His CTCCCCCGTG CCTTCC GTCTGAGTAG GTGTCAT AAG Lys CCT ATC Pro Ile Pme TGA kk CCT Pro GT TTAAACCCGC TGAT AAC CCT Asn Pro TGA CCCTGGAAGG TG TCT ATTCTGGGGG GTGGGGI j TCAGCCT CTC Leu CTC Leu BGH Reverse priming site GGT CTC GAT
5. Early Region Promoter J Molec Appl Gen 1 327 339 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 2009 Life Technologies Corporation All rights reserved 17 Notes 18 invitrogen 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
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7. Val Gln Cys Gly Gly Ile Leu Gln Ile Ser Ser Thr Val V5 epitope I 1801 GGT AAG CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Pme I BGH Reverse priming site il j 1867 CAT TGA GTTTAA ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CAT His 1939 CCCGTGCCTT CCTTGACCCT GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAAT 2019 GAGTAGGTGT CATTCTATTC TGGGGGGTGG GG TTCCATT TCAGGTGTCG TGAGGAATTA 5 end of hEF 1a Exon 2 Kpn BamH Spe I ignal GAG GAAATTGCAT CGCATTGTCT l TA CCG AGC TCG GAT CCA CTA BstB I TIC Phe BstE Il CGA GGT CAC CCA Arg Gly His Pro GAA Glu Polyhistidine region I GGT CAT CAT CAC CAT Gly His His His His CAC His TCTGTTGT TTGCCCCTCC GGGGCAG GACAGCAAGG GGGAGGATTG GGAAGACAAT AGCAGGCATG Transformation and Transfection E coli Transformation a NS NENA Y 4 RECO Nowe Plasmid Preparation Methods of Transfection Positive Control Transform your ligation mixtures into a competent recA endA E coli strain e g TOP10F DH5a and select on LB plates containing 50 100 pg mL ampicillin Select 10 20 clones and analyze for the presence and orientation of your insert We recommend that you sequence your construct with the T7 Forward and BGH Reverse primers sequences to confirm that your gene is fused in frame with the V5 epitope and the C terminal polyhistidine tag Refer
8. for recommendations on sample preparation Human EF 1a Promoter Description Appendix The diagram below shows all the features of the EF 1a promoter used in pEF1 V5 His vectors Mizushima and Nagata 1990 Features are marked as per Uetsuki et al 1989 ame end of human EF la promoter 461 GGAGTGCCTC GTGAGGCTCC GGTGCCCGTC AGTGGGCAGA GCGCACATCG CCCACAGTCC 521 CCGAGAAGTT GGGGGGAGGG GTCGGCAATT GAACCGGTGC CTAGAGAAGG TGGCGCGGGG 581 TAAACTGGGA AAGTGATGTC GTGTACTGGC TCCGCCTTTT TCCCGAGGGT GGGGGAGAAC TATA box Start of Transcription 641 CGTATATAAG TGCAGTAGTC GCCGTGAACG TICTTTTTCG CAACGGGTTT GCCGCCAGAA A Exon I 75 end of Intron 1 TOL CACAGGTAAG TGCCGTGTGT GGTTCCCGCG GGCCTGGCCT CTTTACGGGT TATGGCCCTT 761 GCGTGCCTTG AATTACTTCC ACCTGGCTGC AGTACGTGAT TCTTGATCCC GAGCTTCGGG 821 TTGGAAGTGG GTGGGAGAGT TCGAGGCCTT GCGCTTAAGG AGCCCCTTCG CCTCGTGCTT 881 GAGTTGAGGC CTGGCCTGGG CGCTGGGGCC GCCGCGTGCG AATCTGGTGG CACCTTCGCG 941 CCTGTCTCGC TGCTTTCGAT AAGTCTCTAG CCATTTAAAA TTTTTGATGA CCTGCTGCGA 100 CGCTTTTTTT CTGGCAAGAT AGTCTTGTAA ATGCGGGCCA AGATCTGCAC ACTGGTATTT Sp 1 106 CGGTTTTTGG GGCCGCGGGC_GGCGACGGGG CCCGTGCGTC CCAGCGCACA TGTTCGGCEA Sp 1 112 GGCGGGGECT GCGAGCGCGG CCACCGAGAA TCGGACGGGG GTAGTCTCAA GCTGGCCGGC Sp 1 Sp 1 118 CTGCTCTGGT GCCTGGCCTC GCGCCGCCGT GTATCGCCCC GCCCIEGGGCG GCAAGGCTGG 124 CCCGGTCGGC ACCAGTTGCG TGAGCGGAAA GATGGCCGCT TCCCGGCCCT GCTGCAGGGA Sp 1 130 GCTCAAAATG GAGGACGCGG CGCTCGGGAG AGICGGG
9. to your protein Invitrogen offers the Anti V5 or Anti His C term antibodies to detect your recombinant fusion protein Horseradish peroxidase HRP and alkaline phosphatase AP conjugated antibodies are available for convenient one step detection Antibody Epitope Catalog no Anti V5 Detects a 14 amino acid epitope R960 25 Anti V5 HRP derived from the P and V proteins of R961 25 the paramyxovirus SV5 Southern et al 1991 R962 25 Anti V5 AP GKPIPNPLLGLDST Anti His C term Detects the C terminal polyhistidine R930 25 Anti His C term HRP t28 requires the free carboxyl group R931 25 for detection Lindner et al 1997 Anti His C term AP R932 25 HHHHHH COOH Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e 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
10. 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 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 8148 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 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 Nucleic Acids Res 18 5322 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press 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 Southern P J and Berg P 1982 Transformation of Mammalian Cells to Antibiotic Resistance with a Bacterial Gene Under Control of the SV40
11. TCT Gly Leu Asp Ser 1 ACG CGT ACC GGI CCRC 1 l CGACTGTGCC TTCTAGTTGC CAGCCATCTG 1 rece ACTGTCCTT BGH polyadenylation signal Pro Leu Glu Ser Thr Arg Thr Gly Arg Gly Pro Polyhistidine E T CAT CAT CAC His His His PTGTTTGCCC CCTAATAAAA TGAGGAAAT PT GCATCGCATT GGG GCAGGACAGC AAGGGGGAGG ATTGGGAAGA CAATAGCAGG Continued on next page Cloning into pEF1 V5 His A B and C Continued Multiple Cloning Below is the multiple cloning site for pEF1 V5 His B Restriction sites are Site of Version B labeled to indicate the cleavage site The boxed nucleotides indicate the variable 1579 1659 1734 1800 1866 1939 2019 region The multiple cloning site has been confirmed by sequencing and functional testing For more information on the hEF 1a promoter see page 9 3 end of hEF 1a Intron 1 GTTTGGATCT TGGTTCATTC TCAAGCCTCA GACAGTGGTT CAAAGTTTTT TTCTTCCATT TCAGGTGTCG TGAGGAATTA 5 end of hEF 1a Exon 2 T7 promoter priming site Kpn BamH Spe f 1 GCTTGGTACT AATACGACTC ACTATAGGGA GACCCAAGCT GGCTAGGTAA G CTT GGT ACC GAG CTC GGA TCC ACT Leu Gly Thr Glu Leu Gly Ser Thr BstX EcoR EcoR V BstX Not Xba i I l I l AGT CCA GTG TGG TGG AAT TCT GCA GAT ATC CAG CAC AGT GGC GGC CGC TCG AGT CTA GAG GGC CCG Ser Pro Val Trp Trp Asn Ser Ala Asp Ile Gln His Ser Gly Gly Arg Ser Ser Leu Glu Gly Pro BstB V5 epitope Polyhistidine i r
12. TEGG TGAGTCACCC ACACAAAGGA Ap 1 136 AAAGGGCCTT TCCGTCCTCA GCCGTCGCTT CATGIGACTC CACGGAGTAC CGGGCGCCGT 142 CCAGGCACCT CGATTAGTTC TCGAGCTTTT GGAGTACGTC GTCTTTAGGT TGGGGGGAGG 148 GGTTTTATGC GATGGAGTTT CCCCACACTG AGTGGGTGGA GACTGAAGTT AGGCCAGCTT 154 GGCACTTGAT GTAATTCTCC TTGGAATTTG CCCTTTTTGA GTTTGGATCT TGGTTCATTC 3 end of Intron I 160 TCAAGCCTCA GACAGTGGTT CAAAGTTTTT TTCTTCCATT TCAGGTGTCG TGA 5 end of Exon 2 pEF1 V5 His Vector Map of pEF1 The figure below summarizes the features of the pEF1 V5 His vectors The V5 His sequences for pEF1 V5 His A B and C are available for downloading from 10 www invitrogen com or by contacting Technical Support page 15 Frame dependent variations In Version C there is a unique BstE II site but no Xba site Comments for pEF1 V5 His A 6174 nucleotides EF 1a promoter bases 474 1651 T7 promoter priming site bases 1668 1687 Multiple cloning site bases 1713 1881 V5 epitope bases 1805 1846 Polyhistidine tag bases 1856 1873 BGH reverse priming site bases 1896 1913 BGH polyadenylation sequence bases 1895 2122 f1 origin of replication bases 2172 2600 SV40 promoter and origin bases 2628 2935 Neomycin resistance gene ORF bases 3010 3804 SV40 polyadenylation sequence bases 3980 4110 pUC origin bases 4493 5155 opposite strand Ampicillin resistance gene ORF bases 5300 6160 opposite strand Continued on next page pEF1 V5 His pEF1 V5 His Ve
13. ctor Continued pEF1 V5 His A 6174 bp pEF1 V5 His B 6178 bp and pEF1 V5 His C 6170 bp contain the following elements All features have been functionally tested Feature Benefit Human elongation factor la hEF 1a promoter Allows overexpression of your recombinant protein in a broad range of mammalian cell types Goldman et al 1996 Mizushima and Nagata 1990 T7 promoter priming site Allows for in vitro transcription in the sense orientation and sequencing through the insert Multiple cloning site in three reading frames Allows insertion of your gene and facilitates cloning in frame with the V5 epitope and polyhistidine C terminal tag V5 epitope Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Allows detection of your recombinant protein with the Anti V5 Antibody or Anti V5 HRP Antibody Southern et al 1991 see page 14 for ordering information C terminal polyhistidine tag Allows purification of your recombinant protein on metal chelating resin such as ProBond In addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibody and the Anti His C term HRP Antibody see page 14 for ordering BGH reverse priming site Allows sequencing through the insert Bovine growth hormone BGH Efficient transcription termination and polyadenylation signal polyadenylation of mRNA Goodwin and Rottman 1992 fl origin Allo
14. invitrogen pEF1 V5 His A B and C Catalog no V920 20 Rev date 7 June 2010 Manual part no 28 0184 MAN0000664 ii Table of Contents Kit Contents and Storage nnnnnnsnenensnsenenenenenenenenenenenenenenenenseneneneneneneenenenenenenenenenereeneneneneneneneenenenen iv INTO UCHON tonic 1 Product OvervleW RRA RR 1 o A ee al 2 Cloning into pEF1 V5 His A B and Cisna t i eE e a EE E 2 Transformation and Transfection aans oneens envenvenneenenneenvenvenvenvenvervenneenvenvenvenvenvenvenveenenneenvenvenvenven 6 PADD GT IK see 9 Human EF 1o Promoter nnen voovnrveervserenevservenrenreentennvenneensennoeensnenensounenseonreorventenntennvenseenvenn 9 PEFL V5 His Vectra idad 10 PEFL V5His Medias una seen ae Re dl ene use 12 Accessory Products assor euren ns coed de a AS A eeen glg 13 Technical Support sr eers aandenken knn aankeken bokser beta 15 Purchaser Notification aans ssenre oe vetersnebennesensetdn E RN 16 References muni 17 iii Kit Contents and Storage pEF1 V5 His vectors are shipped on wet ice Upon receipt store vectors at 20 C Shipping and Storage Kit Contents All vectors are supplied as detailed below Store the vectors at 20 C Vector Composition Amount pEF1 V5 His A B and C 40 uL of 0 5 ug uL vector in 10 mM Tris 20 ug HCL 1 mM EDTA pH 8 0 pEF1 V5 His lacZ 40 uL of 0 5 ug uL vector in 10 mM Tris 20 ug HCI 1 mM EDTA pH 8 0 Intended Use For research use
15. line to determine the concentration that kills your cells kill curve Cells differ in their susceptibility to Geneticin Cells will divide once or twice in the presence of lethal doses of Geneticin so the effects of the drug take several days to become apparent Complete selection can take from 3 to 6 weeks of growth in selective medium Continued on next page Transformation and Transfection Continued Preparing Cells for Use the procedure below to prepare cells for lysis prior to purification of your Lysis Lysis of Cells protein on ProBond You will need 5 x 106 to 1 x 107 cells for purification of your protein on a 2 mL ProBond column see the ProBond Purification manual 1 Seed cells in five T 75 flasks or 2 to 3 T 175 flasks 2 Grow the cells in selective medium until they are 80 90 confluent 3 Harvest the cells by treating with trypsin EDTA for 2 to 5 minutes or by scraping the cells in PBS 4 Inactivate the trypsin by diluting with fresh medium if necessary and transfer the cells to a sterile microcentrifuge tube 5 Centrifuge the cells at 240 x g for 5 minutes You may lyse the cells immediately or freeze in liquid nitrogen and store at 80 C until needed TM TM If you are using ProBond resin refer to the ProBond Purification manual for details about sample preparation for chromatography If you are using another metal chelating resin refer to the manufacturer s instruction
16. only Not intended for human or animal diagnostic or therapeutic uses Introduction Product Overview Description of the System Experimental Outline pEF1 V5 His A B and C are 6 2 kb vectors derived from pcDNA 3 1 V5 His and designed for overproduction of recombinant proteins in mammalian cell lines Features of the vectors allow purification and detection of expressed proteins see pages 10 11 for more information High level stable and transient expression can be carried out in most mammalian cells The vectors contain the following elements e Human elongation factor 1a subunit promoter hEF 1a for high level expression across a broad range of species and cell types Goldman et al 1996 Mizushima and Nagata 1990 see page 9 for more information e Three reading frames to facilitate in frame cloning with a C terminal tag encoding the V5 epitope and a polyhistidine metal binding peptide e Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen e g COS7 The control plasmid pEF1 V5 His lacZ is the pEF1 V5 His A vector with a 3 1 kb fragment containing the B galactosidase gene cloned in frame with the C terminal peptide see page 12 It is included for use as a positive control for transfection expression purification and detection in the cell line of choice Use the following outline to clone and express your gene of interest in pEF1 V5 His
17. polyadenylation sequence bases 4899 5126 f1 origin of replication bases 5172 5600 SV40 promoter and origin bases 5654 5936 Neomycin resistance gene ORF bases 6011 6805 SV40 polyadenylation sequence bases 6981 7111 pUC origin bases 7494 8167 opposite strand Ampicillin resistance gene ORF bases 8312 9172 opposite strand Accessory Products Introduction The following products may be used with the pEF1 V5 His vectors For details visit www invitrogen com or contact Technical Support page 15 Item Amount Catalog no 6 x 2 mL precharged prepacked K850 01 ProBond Purification ProBond resin columns and System buffers for native and denaturing purification A 50 mL R801 01 ProBond Resin 150 mL R801 15 Electrocomp TOP10F 5 x 80 uL C665 55 One Shot TOP10F Chemically Competent E coli 20 ALPE u PureLink HiPure Plasmid Miniprep Kit 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 B Gal Assay Kit 80 mL K1455 01 B Gal Staining Kit 1 kit K1465 01 1 gram 11811 023 Geneticin 5 grams 11811 031 25 grams 11811 098 Lipofectamine 2000 Reagent 0 75 mL 11668 027 Primers For your convenience Invitrogen offers a custom primer synthesis service Visit www invitrogen com for more details Continued on next page 13 Accessory Products Continued Antibodies 14 If you do not have an antibody specific
18. r A successful transfection will result in B galactosidase expression that can be easily assayed see next page Continued on next page Transformation and Transfection Continued Assay for B galactosidase Activity Detecting Fusion Proteins Neomycin Geneticin Activity Geneticin Selection Guidelines You may assay for B galactosidase expression by activity assay using cell free lysates Miller 1972 or by staining the cells for activity Invitrogen offers the B Gal Assay Kit and the B Gal Staining Kit for fast and easy detection of B galactosidase expression A number of antibodies are available from Invitrogen that can be used to detect expression of your fusion protein from pEF1 V5 His see page 13 Geneticin blocks protein synthesis in mammalian cells by interfering with ribosomal function It is an aminoglycoside similar in structure to neomycin gentamycin and kanamycin Expression of the bacterial aminoglycoside phosphotransferase gene APH derived from Tn5 in mammalian cells results in detoxification of Geneticin Southern and Berg 1982 Geneticin is available from Invitrogen see page 13 Use as follows e Prepare Geneticin in a buffered solution e g 100 mM HEPES pH 7 3 e Use 100 to 1 000 ug mL of Geneticin in complete medium e Calculate concentration based on the amount of active drug check the lot label e Test varying concentrations of Geneticin on your cell
19. s available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany EF 1alpha 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 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 and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 Goldman L A Cutrone E C Kotenko S V Krause C D and Langer J A 1996 Modifications of Vectors pEF BOS pcDNA1 and pcDNA3 Result in Improved Convenience and Expression BioTechniques 21 1013 1015 Goodwin E C and Rottman
20. 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 please 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 15 Purchaser Notification Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 60 EF 1a Promoter 16 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 Switzerland and is provided only for use in research Information about licenses for commercial use i
21. to the diagrams on pages 3 5 for location and sequence of primer binding sites 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 PureLink HiPure Miniprep Kit or the PureLink HiPure Midiprep Kit see page 13 for ordering information For established cell lines e g HeLa consult original references or the supplier of your cell line for the optimal method of transfection It is recommended 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 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 TM Invitrogen offers the Lipofectamine 2000 Reagent for mammalian transfection pEF1 V5 His lacZ is provided as a positive control vector for mammalian transfection and expression see page 12 It may be used to optimize transfection conditions for your cell line The gene encoding B galactosidase is expressed in mammalian cells under the hEF 1a promote
22. ws rescue of single stranded DNA SV40 early promoter and origin Allows efficient high level expression of the neomycin resistance gene and episomal replication in cells expressing the SV40 large T antigen Neomycin Geneticin resistance gene Selection of stable transfectants in mammalian cells Southern and Berg 1982 SV40 polyadenylation signal Efficient transcription termination and polyadenylation of mRNA pUC origin High copy number replication and growth in E coli Ampicillin resistance gene B lactamase Selection of vector in E coli 11 pEF1 V5 His lacZ Description pEF1 V5 His lacZ is a 9186 bp control vector containing the gene for B galactosidase pEF1 V5 His A was digested with Kpn I and Pme I A 3 1 kb Kpn I Pme I fragment containing the B galactosidase gene was then ligated into the digested pEF1 V5 His A vector in frame with the C terminal peptide Map of Control The figure below summarizes the features of the pEF1 V5 His lacZ vector The Vector nucleotide sequence for pEF1 V5 His lacZ is available for downloading from 12 www invitrogen com or by contacting Technical Support page 15 V5 epitope GxHis frem Comments for pEF1 V5 His lacZ 9186 nucleotides EF 1a promoter bases 470 1653 LacZ portion of the fusion 1721 4777 V5 epitope bases 4805 4846 Polyhistidine tag bases 4856 4873 BGH reverse priming site bases 4896 4913 BGH
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