pUB6/V5-His A, B, and C
Contents
1. Other lysis buffers are suitable 4 Incubate cell suspension at 37 C for 10 minutes to lyse the cells 5 Vortex the cell lysate and centrifuge at 10 000 x g for 10 minutes to pellet nuclei Transfer the supernatant to a fresh tube Assay the supernatant 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 to a final concentration of 1X and boil the sample for 5 minutes 7 Load 20 ug of lysate onto an SDS PAGE gel and electrophorese Use the appropriate percentage of acrylamide to resolve your fusion protein Creating Stable Cell Lines Introduction The pUB6 V5 His vectors contain the blasticidin resistance gene bsd to allow for selection of stable cell lines using blasticidin Kimura et al 1994 We recommend that you test the sensitivity of your mammalian host cell to blasticidin as natural resistance varies among cell lines General information and guidelines are provided below for your convenience Possible Sites for To obtain stable transfectants you may choose to linearize your vector before Linearization transfection While linearizing your vector may not improve the efficiency of transfection it increases the chances that the vector does not integrate in a way that disrupts the gene of interest The table below lists some unique sites that may be used to linearize your const2. Sp 1 AGGGCGCAGG AGCGTCCTGA TCCTT ICCGCC JGGACGCTCA CCAGTATCAG CAGAAGGACA TITTAGGACG GGACTTGGGT GCGAGGAAAA GTAGTCCCTT CTCGGCGATT CTGCGGAGGG Start of Transcription GCGCCGGGTG TGGCACAGCT AGTTCCGTCG CAGCCGGGAT TT 5 end of Intron 1 CTTGGTGAGT AGCGGGCTGC GACCGCCAAG GGCTGTAGTC CTGTTCCCGA GTCTTGAATG GCAAGAACCC AAGGTCTTGA GACCCTGACG TGAAGTTTGT GGGCAGTGCA CCCGTACCTT GTGGGGCCAC CTGCCGGTAG TGAATCGACA GGCGCCGGAC CTTCTTAAGT AGCTGAAGCT TTTTGAAATG TAATCATTTG 3 end of Intron 1 TGGCTTTTTT GTTAGACGAA GGACAGCGGC GACTCTAGGG ATCTCCGTGG CCTCACGGCG CCGCTGCTCA CACTGGTTTT Sp 1 GGCGGTGAAC TTGGGTCGCG GTTCTTGTTT AGCGCTGCCA CGTCAGACGA TAAGACTCGG CCTTAGAACC CTTTCCAGAG AGCGGAACAG TATA box EE GCCGATGATT ATATAAGGAC GTGGATCGCT GTGAT TCGTCA TGGGCTGGCC TGGGTCCGCG GAAGACGCTT GGCCTTCGCT CACTGACTGG TGGGAGCGCG GTGTGCGGTA CTCTGGTGAG CCGGTTTTGA GGTCAATATG GCTTGG 5 end of Exon 2 GGGGCTTTCG AGCAAGGTTG GTGAGGCGGG AATGCGGGAA AGAACTCGGT CGCCCTCGTC GGCTTTTCTC GGGAGGGATA ACTATGCGCT Exon 1 TGGCCGCCGG CCCI CIGI AGCT TIGI TGAACT PGAGG1 TCTTAT TCGTC1 GTG1 IG EG BT IG TCGTGAC CGTCGCAGGA AGTGAGGCGT CGGGGTTGGC GCCGCTCGGT GGGGTTGGGG GTTGAAACAA CGGGTGAGAT TTGCGGGGGC GTCACCCGTT CGCAGGGTTC CAGTTTCTTT GAGTGTGTTT UB Forward priming site f T
3. bases 5553 5608 Blasticidin resistance gene bases 5627 6025 SV40 polyadenylation signal bases 6183 6313 pUC origin bases 6696 7369 Ampicillin resistance gene bases 7514 8374 Blasticidin Description Handling Blasticidin Preparing and Storing Stock Solutions Molecular Weight Formula and Structure 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 Always wear gloves mask goggles and protective clothing e g a laboratory coat when handling blasticidin Weigh out blasticidin and prepare solutions ina hood Blasticidin is soluble in water Water is generally used to prepare stock solutions of 5 to 10 mg mL e Dissolve blasticidin in sterile water and filter sterilize the solution e Aliquot in small volumes suitable for one time use see last point below and freeze at 20 C for long term storage or store at 4 C for short term storage e Aqueous stock solutions are stable for 1 2 weeks at 4 C and 6 8 weeks at 20 C e pH of the aqueous solution shou
4. from a stable cell line 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 1 500 x g for 5 minutes Resuspend the cell pellet in PBS 6 Centrifuge the cells at 1 500 x g for 5 minutes You may lyse the cells immediately or freeze in liquid nitrogen and store 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 metal chelating resin refer to the manufacturer s instruction for recommendations on sample preparation Appendix Human UbC Promoter Description LL 91 17 25 33 41 49 57 65 73 81 89 97 1051 1131 1211 The human UbC promoter allows high level expression of recombinant protein in most mammalian cell lines Wulff et al 1990 and in virtually all tissues tested in transgenic mice Schorpp et al 1996 The diagram below shows the features of the UbC promoter used in pUB6 V5 His Nenoi et al 1996 Features are marked as per Nenoi et al 1996 m 5 end of UbC promoter GAGATCTGGC CTCCGCGCCG GGTTTTGGCG CCTCCCGCGG GCGCCCCCCT
5. B galactosidase Activity Once you have confirmed that your construct is in the correct orientation and fused to the C terminal peptide if desired then you are ready to transfect your cell line of choice We recommend that you include the positive control vector and a mock transfection to evaluate your results 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 TM or the PureLink HiPure Midiprep Kit see page 17 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 1987 Felgner et al 1989 and electroporation Chu et al 1987 Shigekawa and Dower 1988 Invitrogen offers the Lipofectamine 2000 Reagent for mammalian transfection see page 17 for ordering pUB6
6. Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 51 Blasticidin and the Blasticidin Selection Marker 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 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 email outlicensing invitrogen com 19 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 Mol Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nuc Acids Res 15 1311 1326 Fel
7. Note that there are two BstX I sites in the polylinker Transforming Transform your ligation mixtures into a competent recA endA E coli strain Ligation Mixtures e g TOP10F DH5a and select on LB plates containing 50 100 pg mL ampicillin or 50 pg mL blasticidin Select 10 20 clones and analyze for the presence and orientation of your insert ae We recommend that you sequence your construct with the UB Forward and Se BGH Reverse primer sequences to confirm that your gene is fused in frame with TR ST the V5 epitope and the C terminal polyhistidine tag Refer to the diagram on P A pages 3 5 for the sequence and location of the primer binding sites Preparing a Once you have identified the correct clone purify the colony and make a Glycerol Stock glycerol stock for long term storage It is also a good idea to keep a DNA stock of your plasmid at 20 C 1 Streak the original colony out for single colonies on an LB plate containing 50 pg mL ampicillin or 50 g mL blasticidin Incubate the plate at 37 C overnight 2 Isolate a single colony and inoculate into 1 2 mL of LB containing 50 pg mL ampicillin or 50 pg mL blasticidin 3 Grow the culture to mid log phase OD6oo 0 5 0 7 4 Mix 0 85 mL of culture with 0 15 mL of sterile glycerol and transfer to a cryovial 5 Store at 80 C Transfection and Analysis Introduction Plasmid Preparation Methods of Transfection Positive Control Assay for
8. V5 His lacZ is provided as a positive control vector for mammalian transfection and expression see page 14 It may be used to optimize transfection conditions for your cell line The gene encoding B galactosidase is expressed in mammalian cells under the control of the human ubiquitin C hUbC promoter A successful transfection will result in P galactosidase expression that can be easily assayed see below 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 see page 17 for ordering information Continued on next page Transfection and Analysis Continued Detecting Fusion Proteins Several antibodies are available from Invitrogen that can be used to detect expression of your fusion protein from pUB6 V5 His see page 17 for ordering information To detect the fusion protein by western blot you will need to prepare a cell lysate from transfected cells We recommend that you perform a time course to optimize expression of the fusion protein e g 24 48 72 hours etc after transfection To lyse cells 1 Wash cell monolayers 10 cells once with phosphate buffered saline PBS Scrape cells into 1 mL PBS and pellet the cells at 1 500 x g for 5 minutes 3 Resuspend in 50 pL Cell Lysis Buffer see recipe below
9. 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 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 Purchaser Notification Limited Use
10. for selection of stable cell lines see page 15 for more information e Episomal replication in cell lines that are latently infected with SV40 or that express the SV40 large T antigen e g COS 1 COS 7 The control plasmid pUB6 V5 His lacZ is included for use as a positive control for transfection expression and detection in the cell line of choice Use the following outline to clone and express your gene of interest in pUB6 V5 His 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 ug mL ampicillin or 50 ug mL blasticidin Analyze your transformants for the presence of insert by restriction digestion Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in frame with the C terminal peptide 5 Transfect your construct into the cell line of choice Generate a stable cell line if desired 6 Test for expression of your recombinant gene by western blot analysis or other functional assay For antibodies to the V5 epitope or the polyhistidine C terminal tag see the page 17 7 To purify your recombinant protein you may use metal chelating resin such as ProBond ProBond resin is available separately see
11. page 17 for ordering information Methods Cloning into pUB6 V5 His A B and C General Molecular Biology Techniques E coli Strain for Transformation Transformation Method Maintaining pUB6 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 Biology Ausubel et al 1994 Many E coli strains are suitable for the propagation of this vector including TOP10F 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 For your convenience TOP10F is available as chemically competent or electrocompetent cells from Invitrogen see page 17 You may use any method of your choice for transformation Chemical transformation is the most convenient for most researchers Electroporation is the most efficient and the method of choice for large plasmids To propagate and maintain the pUB6 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 or 50 ug mL blasticidin Be
12. sure to prepare a glycerol stock of plasmid containing E coli strain for long term storage see page 5 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 pUB6 V5 His A B and C Continued Multiple Cloning Site of pUB6 V5 His A 1121 1201 1270 1336 1401 1481 Below is the multiple cloning site for pUB6 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 BamH I site and the BstX I site The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence may be downloaded from www invitrogen com or from Technical Support see page 18 For more information on the hUbC promoter see page 11 TTTAGGCACC TTTT TGGCCG
13. AATTTTCAG TGTTAGACTA GTAAATTGTC GGGACGGAAG GGAGCGCAGC GGTGGGGGGC GGGCTGGGGC GGCAGTTATG CTGTTGGCTT GGGCCTAGGG GGTCGGTTTT TGTGAAGTTT CGCTAAATTC CGTGT AAAAT ATGGT ACCAT TGGAGA TGGCGG TGGGCG TCTGGG CGGTGCCGTT ATAATGCAGG TAGGCTCTCC ATGTACCTAT TTTAGGCACC TGGCCGTTTT 11 pUB6 V5 His Vector Map of pUB6 The figure below summarizes the features of the pUB6 V5 His vectors The V5 His sequences for pUB6 V5 His A B and C are available for downloading from 12 www invitrogen com or from Technical Support see page 18 E o V5 epitope 19 oxtiis So 5 o pUB6 V5 His A B C 5 5 kb Comments for pUB6 V5 His A 5463 nucleotides Frame dependent variations BstE Il is only found in version C In addition there are no Xba I UbC promoter bases 18 1227 or Apa I sites in version C UB forward priming site bases 1167 1188 Multiple cloning site bases 1229 1326 V5 epitope bases 1327 1368 Polyhistidine tag bases 1378 1395 BGH reverse priming site bases 1418 1435 BGH polyadenylation signal bases 1421 1648 f1 origin bases 1694 2122 SV40 promoter and origin bases 2149 2458 EM 7 promoter bases 2506 2561 Blasticidin resistance gene bases 2580 2978 SV40 polyadenylation signal bases 3136 3266 pUC origin bases 3649 4322 Ampicillin resistance gene bases 4467 5327 Continued on next page pUB6 V5 His Vector Continued pUB6 V5 His pU
14. B6 V5 His A 5463 bp pUB6 V5 His B 5467 bp and pUB6 V5 His C 5459 bp contain the following elements All features have been functionally tested Feature Benefit Human ubiquitin C hUbC Allows overexpression of your promoter recombinant protein in a broad range of mammalian cell types Hershko and Ciechanover 1982 Wulff et al 1990 Schorpp et al 1996 Multiple cloning site in three reading frames Allows insertion of your gene and facilitates cloning in frame with the V5 epitope and the C terminal polyhistidine 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 Antibodies Southern et al 1991 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 Antibodies Lindner et al 1997 Bovine growth hormone BGH polyadenylation signal Efficient transcription termination and 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 blasticidin resistance gene and episomal replication in cells expressing the SV40 large T antigen EM 7 promoter For expression of the blasticidin resistance gene in E coli B
15. 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 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 Wulff B S O Hare M M Boel E Theill L E and Schwartz T W 1990 Partial Processing of the Neuropeptide Y Precursor in Transfected CHO Cells FEBS Lett 261 101 105 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 2009 Life Technologies Corporation All rights reserved 21 Notes 22 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
16. TTTT TGGC EcoR Pst i I GAA TTC TGC AGA Glu Phe Cys Arg UB forward priming site 1 GAAATG TAATCATTTG GGTCAATATG TAATTTTCAG TGTTAGACTA GTAAATTGTC CGCTAAATTC Hind Ill Asp7181 Kpnl Sac BamH I EcoR V BstX Not I Xho Xba I BstX pol TTTTTT GTTAGAC GAA GCT TGG TAC CGA GCT CGG ATC CAC TAG TCC AGT GTG GTG Glu Ala Trp Tyr Arg Ala Arg Ile His Ser Ser Val Val Apal BstB I I j I SS TAT CCA GCA CAG TGG CGG CCG CTC GAG ICT AGA GGG CCC TTC GAA GGT AAG CCT V5 epitope Age ATC CCT AAC CCT Ile Pro Asn Pro Pme TTAAACCCGC TGAT CCCTGGAAGG TGCC Tyr Pro Ala Gln Trp Arg Pro Leu Glu Ser Arg Gly Pro Phe Glu Gly Lys Pro Polyhistidine region 1 T CTC CTC GGT CTC GAT TCT ACG CGT ACC GGT CAT P CAT CAC CAT CAC CAT TGA GT Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His His His His His BGH reverse priming site 1 CAGCCT CGACTGTGCC TTCTAGTTGC CAGCCATCTG TTGI BGH polyadenylation signal nr 1 ACTCCC ACTGTCCTTT CCTAATAAAA TGAGGAAATT GCAT Note that there are two BstX I sites in the polylinker I TTGCCC CTCCCCCGTG CCTTCCTTGA PCGCATT GTCTGAGTAG GTGTCATTCT Continued on next page Cloning into pUB6 V5 His A B and C Continued Multiple Cloning Below is the multiple cloning site for pUB6 V5 His B Restriction sites are Site of pUB6 V5 labeled to indicate the cleavage site The boxed nucleotides indicat
17. The amount of each reagent provided is listed below Cat no Vector Composition Amount V250 01 pUB6 V5 His A B and C 40 pL of 0 5 ug uL vector in 10 mM Tris 20 ug HCL 1 mM EDTA pH 8 0 pUB6 V5 His lacZ 40 uL of 0 5 ug uL vector in 10 mM Tris 20 ug HCL 1 mM EDTA pH 8 0 Blasticidin powder 50 mg V250 20 pUB6 V5 His A B and C 40 pL of 0 5 ug uL vector in 10 mM Tris 20 ug HCL 1 mM EDTA pH 8 0 pUB6 V5 His lacZ 40 uL of 0 5 ug uL vector in 10 mM Tris 20 ug HCL 1 mM EDTA pH 8 0 Intended Use For research use only Not intended for human or animal diagnostic or therapeutic uses Introduction Product Overview Description of the System Experimental Outline pUB6 V5 His A B and C are 5 5 kb vectors designed for overproduction of recombinant proteins in mammalian cell lines Features of the vectors allow purification and detection of expressed proteins see pages 12 13 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 ubiquitin C promoter hUbC for high level expression across a broad range of species and cell types Schorpp et al 1996 Wulff et al 1990 see page 11 for more information e Three reading frames to facilitate in frame cloning with a C terminal peptide encoding the V5 epitope and a polyhistidine 6xHis metal binding tag e Blasticidin resistance gene bsd
18. e the His B variable region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence may be downloaded from www invitrogen com or from Technical Support see page 18 For more information on the hUbC promoter see page 11 UB forward priming site I 1121 TTTAGGCACC TTTTGAAATG TAATCATTTG GGTCAATATG TAATTTTCAG TGTTAGACTA GTAAATTGTC CGCTAAATTC Hind III Asp7181 Kpnl Sac I BamH BstX I EcoRI l l I I 1201 TGGCCGTTTT TGGCTTTTTT GTTAGACG AAG CTT GGT ACC GAG CTC GGA TCC ACT AGT CCA GTG TGG TGG Lys Leu Gly Thr Glu Leu Gly Ser Thr Ser Pro Val Trp Trp Pst EcoRV BstX I Not Xho Xba I Apa I BstB I I l l l N I 1271 AAT TCT GCA GAT ATC CAG CAC AGT GGC GGC CGC TCG AGT CTA GAG GGC CCG CGG TTC GAA GGT AAG Asn Ser Ala Asp Ile Gln His Ser Gly Gly Arg Ser Ser Leu Glu Gly Pro Arg Phe Glu Gly Lys V5 epitope Age Polyhistidine region 1 I 1 1337 CCT ATC CCT AAC CCT CTC CTC GGT CTC GAT TCT ACG CGT ACC GGT CAT CAT CAC CAT CAC CAT TGA Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His His His His His Pme BGH reverse priming site ij 1 1403 GTTTAAAC CCGCTGATCA GCCTCGACTG TGCCTTCTAG TTGCCAGCCA TCTGTTGTTT GCCCCTCCCC CGTGCCTTCC BGH polyadenylation signal KN 1481 TTGACCCTGG AAGGTGCCAC TCCCACTGTC CTTTCCTAAT AAAATGAGGA AATTGCATCG CATTGTCTGA GTAGGTGTCA Note that there are two BstX I sites in the polylinker Continued on next page Cloning int
19. gner P L Gadek T R Holm M Roman R Chan H W Wenz M Northrop J P Ringold G M and Danielsen M 1987 Lipofectin A Highly Efficient Lipid mediated DNA transfection Procedure Proc Natl Acad Sci USA 84 7413 7417 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 115 121 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 Hershko A and Ciechanover A 1982 Mechanisms of Intracellular Protein Breakdown Ann Rev Biochem 51 335 364 Izumi M Miyazawa H Kamakura T Yamaguchi I Endo 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 Nuc Acids Res 15 8125 8148 Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biol 115 887 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eu
20. ine 2000 Reagent 0 75 mL 11668 027 Electrocomp TOP10F 5 x 80 pL C665 55 One Shot TOP10F 20x50 pL C3030 03 Chemically Competent E coli B Gal Assay Kit 80 mL K1455 01 B Gal Staining Kit 1 kit K1465 01 Antibodies If you do not have an antibody specific 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 t38 requires the free carboxyl group R931 25 for detection Lindner et al 1997 TE Anti His C term AP HHHHHH COOH 932 25 Primers For your convenience Invitrogen offers a custom primer synthesis service Visit www invitrogen com for more details 17 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 Onli
21. invitrogen pUB6 V5 His A B and C Catalog no V250 01 and V250 20 Rev date 30 November 2009 Manual part no 25 0251 MAN0000089 ii Table of Contents Kit Gontents and Storage ranita ea EHER lia iv INrOductian as 1 Product OVERVIEW sanne A A AAA Neen a 1 MEN 008 Sap pios 2 Cloning into pUB6 V5 His A B and C nnunnnenenenenenseneneneneneneesenenesenenennenenenenenenenenensenenenenenennenenen 2 Tr nsfection and An lysisauusssannenkenekskerkaknpadkenknakin iii 6 Creating Stable Cell Lines ikine setes dosa desen E i eeen bedel Ea Eaa dadde sekse Haanen denken 8 PO GTN LI ea eher 11 Human UbC Promoter 2 22 82 erste acuta a a iea STe 11 PUB6 V5 His Vectorial 12 pUBO MOSHIS MAC Lusitania naar AA ad td A A A a GR 14 Blastieidit A RER een en eid 15 Reciper n nenn aa cea god abso ds Saha stien benede gan geeneen idad dabas 16 Accessory Products antennata endt 17 A LG ae adat ack ene sr eases cas daden an dada an kleine 18 Purchaser Notification aats nere ser ae E a NE E TEE E RSE 19 References di EEE 20 iii Kit Contents and Storage Shipping and pUB6 V5 His A B and C vectors are shipped on wet ice Upon receipt store Storage vectors at 20 C Kit Contents The pUB6 V5 His A B and C vectors and the pUB6 V5 His lacZ control plasmid are supplied with each product Cat no V250 01 V250 20 The pUB6 V5 His with Blasticidin Kit Cat no V250 01 also includes blasticidin antibiotic
22. karyotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 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 Nenoi M Mita K Ichimura S Cartwright I L Takahashi E Yamaguchi M and Tsuji H 1996 Heterogeneous Structure of the Polyubiquitin Gene UbC of HeLa S3 Cells Gene 175 179 185 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Edition Plainview New York Cold Spring Harbor Laboratory Press Schorpp M Jager R Schellander K Schenkel J Wagner E F Weiher H and Angel P 1996 The Human Ubiquitin C Promoter Directs High Ubiquitous Expression of Transgenes in Mice Nuc Acids Res 24 1787 1788 Continued on next page 20 References Continued 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
23. lasticidin resistance gene bsd Selection of stable transfectants in mammalian cells Kimura et al 1994 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 13 pUB6 V5 His lacZ Description pUB6 V5 His lacZ is a 8510 bp control vector containing the gene for B galactosidase This vector was constructed by ligating a 3190 bp Hind III Age I fragment containing the lacZ gene and the V5 epitope to a 5320 bp Hind III Age I fragment containing the hUbC promoter polyhistidine tag and blasticidin resistance gene from pUB6 V5 His A Map of Control The figure below summarizes the features of the pUB6 V5 His lacZ vector Vector 14 The nucleotide sequence for pUB6 V5 His lacZ is available for downloading from www invitrogen com or by contacting Technical Support see page 18 o Vs epitope O pUB6 V5 His lacZ 8 5 kb Comments for pUB6 V5 His lacZ 8510 nucleotides UbC promoter bases 18 1227 SV40 pA UB forward priming site bases 1167 1188 LacZ ORF bases 1290 4346 V5 epitope bases 4374 4415 Polyhistidine tag bases 4425 4442 BGH reverse priming site bases 4465 4482 BGH polyadenylation signal bases 4468 4695 f1 origin bases 4741 5169 SV40 promoter and origin bases 5196 5505 EM 7 promoter
24. ld not exceed 7 to prevent inactivation of blasticidin e Do not subject stock solutions to freeze thaw cycles do not store in a frost free freezer e Upon thawing use what you need and discard the unused portion The formula for blasticidin is C17H gt sN3O5 HCI and the molecular weight is 458 9 The diagram below shows the structure of blasticidin NH2 SEN Ps HOOC O CH3 HCI DAA NH NH2 O 15 Recipe Cell Lysis Buffer 16 50 mM Tris 150 mM NaCl 1 Nonidet P 40 pH 7 8 1 3 This solution can be prepared from the following common stock solutions For 100 mL combine 1M Tris base 5 mL 5M NaCl 3mL Nonidet P 40 1 mL Bring the volume to 90 mL with deionized water and adjust the pH to 7 8 with HCI Bring the volume up to 100 mL Store at room temperature Note Protease inhibitors may be added at the following concentrations 1 mM PMSF 1 pg mL Pepstatin 1 pg mL Leupeptin Accessory Products Introduction The following products may be used with the pUB6 V5 His vectors For details visit www invitrogen com or contact Technical Support page 18 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 50 mL R801 01 ProBond Resin 150 mL R801 15 PureLink HiPure Plasmid 25 preps K2100 04 Midiprep Kit Lipofectam
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26. o pUB6 V5 His A B and C Continued Multiple Cloning Below is the multiple cloning site for pUB6 V5 His C Restriction sites are Site of pUB6 V5 labeled to indicate the cleavage site The boxed nucleotides indicate the His C variable region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence may be downloaded from www invitrogen com or from Technical Support see page 18 UB forward priming site 1121 TTTAGGCACC TTTTGAAATG TAATCATTTG GGTCAATATG TAATTTTCAG TGTTAGACTA GTAAATTGTC CGCTAAATTC Hind III Asp7181 Kpnl Sac I BamH BstX I EcoR I 1201 TGGCCGTTTT TGGCTTTTTT GTTAGACGA AGC TTG GTA CCG AGC TCG GAT CCA CTA GTC CAG TGT GGT GGA Ser Leu Val Pro Ser Ser Asp Pro Leu Val Gln Cys Gly Gly Pst EcoRV Bstx Not Xho I BstE II BstB V5 epitope 1272 ATT CTG CAG ATA TCC AGC ACA GTG GCG GCC GCT CGA GET CAC CCA TTC GAA GGT AAG CCT ATC CCT Ile Leu Gln Ile Ser Ser Thr Val Ala Ala Ala Arg Gly His Pro Phe Glu Gly Lys Pro Ile Pro Age Polyhistidine region Pme I 1338 AAC CCT CTC CTC GGT CTC GAT TCT ACG CGT acc GGT CAT CAT CAC CAT CAC CAT TGA GTTTAA Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly His His His His His His BGH Reverse priming site T 1 1401 ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TTGCCCCTCC CCCGTGCCTT CCTTGACCCT BGH polyadenylation signal 1481 GGAAGGTGCC ACTCCCACTG TCCTTTCCTA ATAAAATGAG GAAATTGCAT CGCATTGTCT GAGTAGGTGT CATTCTATTC
27. ruct prior to transformation Other restriction sites are possible Note that for the enzymes listed below the cleavage site is indicated for versions A B and C of pUB6 V5 His Be sure that your insert does not contain the restriction enzyme site you wish to use to linearize your vector Enzyme Location Supplier Bel II Upstream of hUbC Many promoter Bst1107 I End of SV40 polyA AGS Fermentas Takara Sap 1 Backbone New England Biolabs BspLU 111 Backbone Boehringer Mannheim AlwN I pMBI origin Amersham New England Biolabs Life Technologies Eam1105 I Ampicillin gene AGS Fermentas Takara Bell Ampicillin gene Many Fsp 1 Ampicillin gene Many Sca I Ampicillin gene Many Ssp I Backbone Many Continued on next page Creating Stable Cell Lines Continued Selection in Mammalian Cell Lines Selecting Stable Integrants To generate a stable cell line expressing your protein you need to determine the minimum concentration of blasticidin required to kill your untransfected host cell line Typically concentrations between 2 and 10 pg mL blasticidin are sufficient to kill the untransfected host cell line Test a range of concentrations see below to ensure that you determine the minimum concentration necessary for your cell line See page 15 for details on handling and preparing Blasticidin solution L Seed cells at 20 25 confluency for each time point 6 time points and allo
28. w the cells to adhere overnight The next day substitute culture medium with medium containing varying concentrations of blasticidin e g 0 1 3 5 7 5 and 10 ug mL blasticidin Replenish the selective medium every 3 4 days Cells sensitive to blasticidin will round up and detach from the plate Dead cells will accumulate in the medium Count the number of viable cells at regular intervals to determine the appropriate concentration of blasticidin that prevents growth Once the appropriate concentration of blasticidin is determined you can generate a stable cell line with your construct 1 Transfect your cells using the optimal protocol for your cell line Include a sample of untransfected cells as a negative control 48 hours after transfection split the cells into fresh medium containing blasticidin at the appropriate concentration for your cell line Split the cells such that they are no more than 25 confluent Replenish selective medium every 34 days until blasticidin resistant colonies are detected Pick and expand colonies Continued on next page Creating Stable Cell Lines Continued Preparing Cells for Lysis Lysis of Cells 10 Use the procedure below to prepare cells for lysis prior to purification of your protein on ProBond You will need 5 x 10 to 1 x 107 cells for purification of your protein on a 2 mL ProBond column see ProBond Protein Purification manual 1 Seed cells
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