pSecTag2/Hygro Vector
Contents
1. Shigekawa K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells Bio Techniques 6 742 751 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 1997 2009 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 14 Notes Notes invitrogen Corporate Headquarters Invitrogen Corporation 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 com2. 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 complete medium Transfer the cells to a sterile conical tube 5 Centrifuge the cells at 1500 rpm for 5 minutes You may wish to wash the cells one time with PBS prior to lysis The cells may be lysed immediately or frozen in liquid nitrogen and stored at 80 C until needed If you are using ProBond resin refer to the ProBond Protein Purification manual for details about sample preparation for chromatography If you are using other resin refer to the manufacturer s instruction for recommendations on sample preparation Appendix pSecTag2 Hygro PSA Description pSecTag2 Hygro PSA is a 6408 bp positive control vector expressing and Map secreting the prostate specific antigen PSA fused to the c myc epitope and the polyhistidine tag The vector was constructed by amplifying the PSA gene and cloning it into pCR I The fragment of DNA containing the PSA gene was excised using Kpn I and Apa I and cloned into Kpn 1 Apa I digested pSecTag2 Hygro A The figure below shows the features of pSecTag2 Hygro PSA The complete nucleotide sequence for pSecTag2 Hygro PSA is available for downloading from our Web site www invitrogen com or from Technical Service page 13 T7 4 ATG lgx Leader myc epitope
3. 6xHis TAA pSecTag2 Hygro PSA Comments for pSecTag2 Hygro PSA 6408 nucleotides The Sfi I site is located before the variable region CMV promoter bases 209 863 T7 promoter priming site bases 863 882 Murine lg kappa chain V J2 C signal peptide bases 905 967 PSA gene bases 1042 1732 c myc epitope bases 1745 1777 Polyhistidine tag bases 1790 1807 BGH reverse priming site bases 1830 1847 BGH polyadenylation sequence bases 1829 2043 f1 origin bases 1954 2367 SV40 promoter and origin bases 2587 2908 Hygromycin B phosphotransferase ORF Hyg bases 2926 3951 SV40 polyadenylation site bases 4144 4210 pUC origin bases 4594 5267 B lactamase ORF Amp bases 5412 6272 11 Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes MSDSs 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 Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business P
4. Ie Ser Glu Glu Asp Leu Allows detection of pSecTag2 Hygro fusion protein with the Anti myc Antibody Catalog no R950 25 Evans et al 1985 Polyhistidine tag For high affinity binding to Ni chelating resin i e ProBond and easy purification In addition it allows detection of pSecTag2 Hygro fusion proteins with the Anti His C term Antibody Catalog no R930 25 Lindner et al 1997 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 Allows rescue of single stranded DNA SV40 early promoter and origin Allows efficient high level expression of the hygromycin resistance gene and episomal replication in cells expressing the SV40 large T antigen Hygromycin resistance gene Hygromycin B phosphotransferase Selection of stable transfectants in mammalian cells Gritz and Davies 1983 Palmer et al 1987 SV40 polyadenylation signal Efficient transcription termination and polyadenylation of mRNA pUC origin High copy number replication and growth in E coli Ampicillin resistance gene lactamase Selection in E coli continued on next page pSecTag2 Hygro Vector continued Map of The figure below shows where the features of pSecTag2 Hygro are located in pSecTag2 Hygro the ve
5. TATA 3 end ofh CMV Fn PP 1 759 GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT putative transcriptional start T7 promoter primer binding site I 819 CTCTGGCTAA CTAGAGAACC CACTGCTTAC TGGCTTATCG AAATTAATAC GACTCACTAT Ig k chain leader sequence l 879 AGGGAGACCC AAGCTGGCTA GCCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG Met Glu Thr Asp Thr Leu Leu Leu Trp S I 1 932 GTA CTG CTG CTC TGG GTT CCA GGT TCC ACT GGT GAC GCG GCC CAGCCG Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Asp Signal cleavage site Hind Il Asp7181 KpnI BamH I Bst Ei I I 980 GCCAGGCGC GCGCGCCG TACGAAG CTTGGTACCG AGCTCGGATC CACTCCAGTG TGGTGGA EcoR V BstX1 NotI XhoI Drail Apal l I l 1041 ATTCTGCAGA TATCCAGCAC AGTGGCGGCC GCTCGAGGAG GGCCC GAA CAA AAA CTC Glu Gln Lys Leu myc epitope Polyhistidine tag m 1098 ATC TCA GAA GAG GAT CTG AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT Ile Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His BGH reverse priming site 1 1146 CAT TGA GTTTAAACCC GCTGATCAGC CTCGACTGTG CCTTCTAGTT GCCAGCCATC His KKK 1202 TGTTGTTTGC CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT BGH poly A addition site Pl 1262 TTCCTAATAA AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGT continued on next page Cloning into pSecTag2 Hygro continued Multiple Cl Site of pSecTag2 Hygro C 699 oning Below is the mult
6. in the correct reading frame prepare 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 DNA using the PureLink HiPure Plasmid MiniPrep or Plasmid MidiPrep kits or the ChargeSwitch Pro Plasmid Miniprep Kit see page vi Additional Products Transfection into Mammalian Cells Methods of Transfection Hygromycin B Selection Guidelines Hygromycin B Activity 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 If you wish to create stable cell lines expressing your gene of interest you will need to select using hygromycin B Use hygromycin B 527 5 MW as follows e Test varying concentrations of hygromycin on your cell line to determine the concentration that kills your cells kill curve Cells differ in their susceptibility to hygromycin e Prepare complete medium supplemented with 100 to 1000 pg ml hygromycin B Generally concentrations that kil
7. sequence 1 M 879 AGGGAGACCC AAGCTGGCTA GCCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG Met Glu Thr Asp Thr Leu Leu Leu Trp Sfi l 1 I 932 GTA CTG CTG CTC TGG GTT CCA GGT TCC ACT GGT GAC GCG GCC CAGCCG Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly ASP Signal cleavage site Asc I Hind UI Asp7181 KpnI BamH I en I I 980 GCCAGGCGC GCCG TACGAAG CTTGGTACCG AGCTCGGATC CACTCCAGTG TGGTGGAATT EnEV BstX I oy anol Drall Apal myc epitope l Ie 1040 CTGCAGATAT CCAGCACAGT GGCGGCCGCT CGAGGAGGGC CC GAA CAA AAA CTC ATC Glu Gln Lys Leu Ile Polyhistidine tag 1097 TCA GAA GAG GAT CTG AAT AGC GCC GTC GAC CAT CAT CAT CAT CAT CAT TGA Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His His His His BGH reverse priming site De venne 1148 GTTTAAACCC GCTGATCAGC CTCGACTGTG CCTTCTAGTT GCCAGCCATC TGTTGTTTGC BGH poly A addition site 1208 CCCTCCCCCG TGCCTTCCTT GACCCTGGAA GGTGCCACTC CCACTGTCCT TTCCTAATAA ml 1268 AATGAGGAAA TTGCATCGCA TTGTCTGAGT AGGTGT continued on next page Cloning into pSecTag2 Hygro continued Multiple Cloning Below is the multiple cloning site for pSecTag2 Hygro B Restriction sites are Site of labeled to indicate the cleavage site The variable region is the boxed region pSecTag2 located after the Ig kappa chain leader sequence The multiple cloning site has Hygro B been confirmed by sequencing and functional testing enhancer region 3 end 699 AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAACAACTCC CAAT
8. 15 121 Felgner P L 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 Gritz L and Davies J 1983 Plasmid Encoded Hygromycin B Resistance The Sequence of Hygromycin B Phosphotransferase Gene and its Expression in E coli and 5 Cerevisiae Gene 25 179 188 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 Bio Techniques 22 140 149 Nelson J A Reynolds Kohler C and Smith B A 1987 Negative and Positive Regulation by a Short Segment in the 5 Flanking Region of the Human Cytomegalovirus Major Immediate Early Gene Molec Cell Biol 7 4125 4129 Palmer T D Hock R A Osborne W R A and Miller A D 1987 Efficient Retrovirus Mediated Transfer and Expression of a Human Adenosine Deaminase Gene in Diploid Skin Fibroblasts from an Adenosine Deficient Human Proc Natl Acad Sci U S A 84 1055 1059 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press
9. A GAA GAG GAT CTG AAT AGC GCC GTC GAC CAT CAT CAT le Ser Glu Glu Asp Leu Asn Ser Ala Val Asp His His His BGH reverse priming site CCTT GACCCTGGAA GGTGCCACTC CGCA TTGTCTGAGT AGGTGT continued on next page Transformation into E coli Introduction E coli Transformation N N MENO 7 RECO Nowe l Plasmid Preparation At this point you should have ligation mixtures that are ready to be transformed into competent E coli The following guidelines and recommendations are provided for your convenience If you need more details about the techniques discussed refer to the general molecular biology references in the Reference section Transform your ligation mixtures into a competent recA endA E coli strain e g INVaF 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 17 forward and BGH reverse primer binding sites to confirm that your gene is correctly fused to the Ig kappa chain leader sequence at the N terminal and the C terminal tag Refer to diagrams on pages 5 7 for sequence and location of primer binding sites For your convenience Invitrogen offers a custom primer synthesis service For more information refer to our website www invitrogen com or contact Technical Support page 13 Once you have confirmed that your gene is
10. Andersson S Davis D L Dahlb ck H J rnvall H and Russell D W 1989 Cloning Structure and Expression of the Mitochondrial Cytochrome P 450 Sterol 26 Hydroxylase a Bile Acid Biosynthetic Enzyme J Biol Chem 264 8222 8229 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 Boshart M Weber F Jahn G Dorsch H sler K Fleckenstein B and Schaffner W 1985 A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus Cell 41 521 530 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 Coloma M J Hastings A Wims L A and Morrison S L 1992 Novel Vectors for the Expression of Antibody Molecules Using Variable Regions Generated by Polymerase Chain Reaction J Imm Methods 152 89 104 Evans G I Lewis G K Ramsay G and Bishop V M 1985 Isolation of Monoclonal Antibodies Specific for c myc Proto oncogene Product Mol Cell Biol 5 3610 3616 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 1
11. ark Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 ORF 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 techsupport invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Certificate of Analysis Limited Warranty 12 Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds The Certificate of Analysis CofA provides detailed quality control information for each product The CofA is available on our website at www invitrogen com cofa and is searchable by product lot number which is printed on each box Invitrogen 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 Invitrogen warrants that all of its products will 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 Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expirati
12. ctor The sequences for pSecTag2 Hygro A B and C are available for downloading from our Web site www invitrogen com or from Technical Service page 13 Details of the multiple cloning sites for all three vectors including the variable region that determines the reading frame are shown on pages 5 7 ATG Igx Leader myc epitope 6xHis TAA pSecTag2 Hygro A B C The Sfi I site is located before the variable region t The Asc is only found in Comments for pSecTag2 Hygro A version A 5745 nucleotides CMV promoter bases 209 863 T7 promoter priming site bases 863 882 Murine lg kappa chain V J2 C signal peptide bases 905 967 Multiple cloning site bases 970 1081 c myc epitope bases 1082 1111 Polyhistidine tag bases 1127 1144 BGH reverse priming site bases 1167 1184 BGH polyadenylation site bases 1166 1380 f1 origin bases 1443 1856 SV40 promoter and origin bases 1924 2245 Hygromycin B phosphotransferase ORF Hyg bases 2263 3288 SV40 polyadenylation site bases 3418 3547 pUC origin bases 3931 4604 B lactamase ORF Amp bases 4749 5609 Methods Cloning into pSecTag2 Hygro Introduction General Molecular Biology Techniques Maintenance of pSecTag2 Hygro Cloning into the pSecTag2 Hygro Vectors Note This section contains information on cloning your insert into the pSecTag2 Hygro vectors Details of the multiple cloning sites are found on pages 5 7 Brief information on tra
13. d MiniPrep Kits 25 preps K2100 02 100 preps K2100 03 PureLink HiPure Plasmid MidiPrep Kits 25 preps K2100 04 50 preps K2100 05 ChargeSwitch Pro Plasmid Miniprep Kit 50 preps C530050 250 preps C530250 Anti myc Antibody 25 westerns R950 25 Anti His C term Antibody 25 westerns R930 25 ProBond Purification System 6 K850 01 purifications ProBond Metal Binding Resin 50 ml R801 01 150 ml R801 15 Purification Columns 10 ml 50 R640 50 polypropylene columns Overview Introduction E coli Strain Introduction pSecTag2 Hygro A B and C are 5 7 kb expression vectors designed for high level expression and secretion in mammalian hosts The pSecTag2 Hygro vectors are identical to the pSecTag2 vectors except that the Zeocin resistance gene is replaced with the hygromycin B resistance gene Gritz and Davies 1983 for selection in mammalian cells Like pSecTag2 pSecTag2 Hygro contains the gene encoding P lactamase for bacterial selection on ampicillin Proteins expressed from pSecTag2 Hygro are fused at the N terminus to the murine Ig kappa chain leader sequence for protein secretion and at the C terminus to a peptide containing the c myc epitope and six tandem histidine residues for detection and purification The pSecTag2 Hygro vector is supplied in three different versions A B and C to facilitate correct in frame fusion with the Ig kappa chain leader sequence For more information on the pSecTag2 H
14. for secreted recombinant protein by functional assay or western blot analysis It may be necessary to perform a time course analysis to determine the optimal time for expression of your recombinant protein If you do not have an antibody to your particular protein you can use the Anti myc Antibody to detect your protein A positive control vector pSecTag2 Hygro PSA is included to test for expression and secretion in your particular cell line Prostate specific antigen PSA is fused to the c myc epitope and the polyhistidine tag The resulting fusion protein is 33 kDa which includes the N terminal secretion signal When the secretion signal is cleaved off the size of the fusion protein should decrease by 2 4 kDa Note There are glycosylation sites in PSA The secreted fusion protein expressed in human breast carcinoma cells migrates at 45 kDa To purify secreted recombinant protein from the medium follow the manufacturer s instructions for the nickel chelating resin that you are using Start with about 3 to 5 ml of medium and load onto 1 to 2 ml of resin Scale up or down depending on the level of expression If you do not detect any secreted protein in the medium use the procedure below to check cells for production of recombinant protein You will need 5 x 10 to 1 x 107 cells for purification on a 2 ml ProBond column see ProBond Protein Purification manual 1 Seed cells in either five 1 75 flasks or 2 to 3 T 175 flasks
15. invitrogen pSecTag2 Hygro A B and C Catalog no V910 20 Version E ii Table of Contents KitCont nts and ro ae nee v Additonal F dt As vi lg A neten seated detoneren dt 1 OR EE 1 Peder Hygro VEO EA A AAA AER A 2 A EE acstaactaahtacsiadasdennacatdanigSaisanay Snsdantetacads 4 Cloning into poet hag27 EVO nde 4 Transformatoninto ESO Jensen daneen 8 Transfection into Mammalian Cells se 9 Expression ad Greer 10 APBENGN Cuna ted 11 Ped STO ESA seeden 11 Technical SUP DO A Beate be neen 12 Purchaser NOA CAOS A eneen 13 References un een 14 iii iv Kit Contents and Storage Kit Contents Shipping and Storage Product Qualification Each kit contains 20 ug each of pSecTag2 Hygro A B and C and pSecTag2 Hygro PSA Each vector is supplied at a concentration of 0 5 ug pl in 40 ul of 10 mM Tris HCI 1 mM EDTA pH 8 0 Plasmids are shipped on ice and should be stored at 20 C The Certificate of Analysis CofA provides detailed quality control information for each product The CofA is available on our website at www invitrogen com cofa and is searchable by product lot number which is printed on each box Additional Products Kit Contents vi The following products are available separately from Invitrogen To order visit our website at www invitrogen com or contact Technical Support see page 13 Product Amount Catalog number PureLink HiPure Plasmi
16. iple cloning site for pSecTag2 Hygro C Restriction sites are labeled to indicate the cleavage site The variable region is the boxed region located after the Ig kappa chain leader sequence The multiple cloning site has been confirmed by sequencing and functional testing enhancer region 3 end AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACI CAAT AL TTC CAAAATGTCG TAACAACTCC TATA 3 end of hCMV mars 759 GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT pe T7 promoter primer binding site 819 CTCTGGCTAA CTAGAGAACC CACTGCTTAC TGGCTTATCG AAATTAATAC GACTCACTAT Ig k chain leader sequence Fe 879 AGGGAGACCC AAGCTGGCTA GCCACC ATG GAG ACA GAC ACA CTC CTG CTA TGG Met Glu Thr Asp Thr Leu Leu Leu Trp S I 932 GTA CTG CTG CTC TGG GTT CCA GGT TCC ACT GGT GAC GCG GCC CAGCCG Val Leu Leu Leu Trp Val Pro Gly Ser Thr GlyyAsp Signal cleavage site Hind Wl Asp7181 KpnI BamHI I I I 980 GCCAGGCGC GCGCGCCGTACG TACGAAG CTTGGTACCG AGCTCGGATC CACTCCAGTG as Bek v Buel Noon Dra II Apal 1038 TGGTGGAATT CTGCAGATAT CCAGCACAGT GGCGGCCGCT CGAGGAGGGC CC GAA CAA Glu Gln myc epitope Polyhistidine tag TE m 1096 1144 1196 1256 AAA CTC Al Lys Leu I BGH poly A addition site m CAT CAT CAT TGA GTTTAAACCC GCTGATCAGC CTCGACTGTG CCTTCTAGTT His His His GCCAGCCATC TGTTGTTTGC CCCTCCCCCG TGCCTI IT CCACTGTCCT TTCCTAATAA AATGAGGAAA TTGCAT TC TC
17. l mammalian cells are in the 150 to 400 pg ml range e Calculate concentration based on the amount of active drug check the lot label Cells will divide once or twice in the presence of lethal doses of hygromycin so the effects of the drug take several days to become apparent Complete inhibition of cell growth can take 2 to 3 weeks of growth in selective medium Selection and expansion of clones will take additional time Hygromycin B Catalog no 10687 010 is an aminocyclitol that inhibits protein synthesis by disrupting translocation and promoting mistranslation It is used for selection of stable mammalian cell lines The resistance gene encodes hygromycin B phosphotransferase which detoxifies hygromycin B by phosphorylation Expression and Purification Introduction Detection of Secreted Protein from Medium Purification of Secreted Recombinant Protein Analyzing Cells for Recombinant Protein Lysis of Cells 10 Expression of your recombinant protein can be detected using an antibody to the c myc epitope encoded in the C terminal fusion peptide In addition the metal binding domain allows simple one step purification of your recombinant protein by Immobilized Metal Affinity Chromatography IMAC using a nickel chelating resin i e ProBond See Additional Products on page vi for detection and purification products from Invitrogen The medium in which transfected cells are grown can be analyzed
18. nsforming into E coli is located below For help with DNA ligations E coli transformations restriction enzyme analysis purification of single stranded DNA DNA sequencing and DNA biochemistry see Molecular Cloning A Laboratory Manual Sambrook et al 1989 or Current Protocols in Molecular Biology Ausubel et al 1994 To propagate and maintain pSecTag2 Hygro A B and C we recommend that you transform the plasmids into E coli and prepare glycerol stocks for long term storage as described below 1 Use the supplied 0 5 ug ul stock solution in TE pH 8 0 to transform a recA endA E coli strain like INVaF TOP10F DH5aF or equivalent Select transformants on LB plates containing 50 100 pg ml ampicillin 3 Analyze transformants for the appropriate plasmid and prepare glycerol stocks by mixing 0 85 ml of an overnight culture with 0 15 ml of sterile glycerol Transfer the resulting solution to a cryovial and store at 80 C pSecTag2 Hygro A B and C vectors are fusion vectors requiring that you clone your gene of interest in frame with the initiation ATG of the N terminal Ig kappa chain leader sequence and or the C terminal myc epitope polyhistidine tag Three versions of this vector are provided to facilitate cloning For proper expression first determine which restriction sites are appropriate for ligation and then which vector will preserve the reading frame at BOTH the 5 and the 3 ends It may be necessa
19. on date No warranty is applicable unless all product components are stored in accordance with instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen 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 Invitrogen 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 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 Purchaser Notification Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer 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 is available from QIAGEN GmbH Max Volmer Str 4 D 40724 Hilden Germany 13 References
20. ry to PCR your gene product to create a fragment with the appropriate restriction sites to clone in frame at both ends Carefully inspect your gene and the multiple cloning site of each vector before cloning your gene of interest Be sure to remove the stop codon in your gene if you wish to express your protein with the C terminal tag See pages 5 7 for details of the multiple cloning sites If you wish to express and secrete your protein without the C terminal tag include a termination codon in your gene of interest Note that you will be unable to detect the fusion protein with the Anti myc Antibody or Anti His C term Antibody or purify it using nickel chelating resin i e ProBond continued on next page Cloning into pSecTag2 Hygro continued Multiple Cloning Below is the multiple cloning site for pSecTag2 Hygro A Restriction sites are Site of labeled to indicate the cleavage site The variable region is the boxed region pSecTag2 een a re kappa chain Bee a Kr multiple cloning site has een contirme sequencing and runctuonal testing enhancer region 3 end 699 AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG TAACAACTCC CAAT TATA 3 end of hCMV 1 P 1 759 GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT AAGCAGAGCT tha dd siart T7 promoter primer binding site l 819 CTCTGGCTAA CTAGAGAACC CACTGCTTAC TGGCTTATCG AAATTAATAC GACTCACTAT Ig k chain leader
21. ygro vector see page 2 To get started with cloning into pSecTag2 Hygro see page 4 We recommend that you propagate pSecTag2 Hygro A Band C in E coli strains that are recombination deficient recA and endonuclease A deficient endA such as TOP10F DH5aF and INVaF For your convenience TOP10F is available as chemically competent or electrocompetent cells from Invitrogen Item Quantity Catalog no Electrocomp TOP10F 5 x 80 ul C665 55 One Shot TOP10F chemically competent cells 21 x 50 pl C3030 03 pSecTag2 Hygro Vector Features of pSecTag2 Hygro A 5745 bp pSecTag2 Hygro B 5749 bp and pSecTag2 Hygro pSecTag2 Hygro C 5753 bp contain the following elements All features have been functionally tested Feature Benefit Human cytomegalovirus CMV immediate early promoter enhancer Allows efficient high level expression of your recombinant protein Andersson et al 1989 Boshart et al 1985 Nelson et al 1987 T7 promoter priming site Allows for in vitro transcription in the sense orientation and sequencing through the insert ATG initiation codon Allows initiation of translation of the pSecTag2 Hygro fusion protein Murine Ig k chain leader sequence Allows secretion of the fusion protein Coloma et al 1992 Multiple cloning site Allows insertion of your gene and facilitates cloning c myc epitope Glu Gln Lys Leu
Download Pdf Manuals
Related Search