pcDNA - Molecular Info
Contents
1. Item Amount Catalog no 6 x 2 mL precharged prepacked K850 01 ProBond Purification System nen ken Oen buffers for native and denaturing purification i 50 mL R801 01 ProBond Resin 150 mL R801 15 Anti Xpress Antibody 50 uL R910 25 Electrocomp TOP10F 5x80 uL C665 55 One Shot TOP10F 21x50 ul C3030 03 chemically competent cells 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 R250 01 Zeocin 5 gram R250 05 Lipofectamine 2000 Reagent 0 75 mL 11668 027 EnterokinaseMax 250 units E180 01 For your convenience Invitrogen offers a custom primer synthesis service Visit www invitrogen com for more details 19 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 5791 Van Allen Way LOOP X Bldg2. chelating resin such as ProBond Xpress epitope tag Allow detection of your recombinant protein with the Anti Xpress Antibody Enterokinase cleavage site Allows removal of the N terminal tag from your recombinant protein using an enterokinase such as TM EnterokinaseMax Multiple cloning site in three reading frames Allows insertion of your gene and facilitates cloning in frame with the Xpress epitope and N terminal polyhistidine tag BGH reverse priming site Permits sequencing through the insert 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 Zeocin resistance gene in mammalian cells and episomal replication in cells expressing the SV40 large T antigen EM 7 promoter Synthetic promoter based on the bacteriophage T7 promoter for expression of the Zeocin resistance gene in E coli Zeocin resistance gene Selection of transformants in E coli and stable transfectants in mammalian cells Drocourt et al 1990 Mulsant et al 1988 SV40 polyadenylation signal Efficient transcription termination and polyadenylation of mRNA pUC origin High copy number replication and growth in E coli Ampicillin resistance
3. 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech supportQinvitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Certificate of Analysis Limited Warranty 20 Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of charge any product that does not meet those specifications This warranty limits the Company s li
4. Introduction Zeocin Molecular Weight Formula and Structure 16 The pcDNA 4 HisMax vectors contain the Zeocin resistance gene for selection of stable cell lines using Zeocin We recommend that you test the sensitivity of your mammalian host cell to Zeocin as natural resistance varies among cell lines General information and guidelines are provided in this section for your convenience Zeocin is a member of the bleomycin phleomycin family of antibiotics isolated from Streptomyces Antibiotics in this family are broad spectrum antibiotics that act as strong anti bacterial and anti tumor drugs They show strong toxicity against bacteria fungi including yeast plants and mammalian cells Baron et al 1992 Drocourt et al 1990 Mulsant et al 1988 Perez et al 1989 The Zeocin resistance protein has been isolated and characterized Calmels et al 1991 Drocourt et al 1990 This protein the product of the Sh ble gene Streptoalloteichus hindustanus bleomycin gene is a 13 7 kDa protein that binds Zeocin and inhibits its DNA strand cleavage activity Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin The formula for Zeocin is Cz5HsO N20S2Cu HCI and the molecular weight is 1 527 5 daltons Zeocin is an HCI salt The diagram below shows the structure of TM Zeocin Continued on next page Zeocin Continued Applications of Z
5. 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 Invitrogen offers the Lipofectamine 2000 Reagent for mammalian transfection Refer to our website www invitrogen com or call Technical Support see page 20 for more information TM pcDNA 4 HisMax lacZ is provided as a positive control vector for mammalian cell transfection and expression see page 15 and 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 CMV promoter A successful transfection will result in B 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 19 Continued on next page Transfection and Analysis Continued Detecting Fusion Proteins Note Purification The Anti Xpress antibodies and the Anti HisG antibodies are available from Invitrogen to detect expression of your fusion protein from pcDNA 4 HisMax see page 19 To detect the fusion protein by western blot you need to prepar
6. 1 1251 ACCCGCTGAT CAGCCTCGAC TGTGCCTTCT AGTTGCCAGC CATCTGTTGT TIGCCCCTCC Cloning into pcDNA 4 HisMax A B and C Continued E coli Transformation Important N SENO 7 O RE NS I Preparing a Glycerol Stock 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 or 25 50 ug mL Zeocin in Low Salt LB medium see page 18 Select 10 20 clones and analyze for the presence and orientation of your insert Any E coli strain that contains the complete Tn5 transposable element i e DH5aFTO SURE SURE2 encodes the ble gene bleomycin resistance gene These strains will confer resistance to Zeocin For the most efficient selection we recommend an E coli strain that does not contain the Tn5 gene i e TOP10 DH5a DH10 etc We recommend that you sequence your construct with the T7 Forward and BGH Reverse primers to confirm that your gene is fused in frame with the N terminal polyhistidine tag and the Xpress epitope For ordering primers see page 19 Note that if you use the T7 Forward primer to sequence your insert approximately 300 bp of sequence encoding the QBI SP163 element and the N terminal tag will precede the sequence of your insert Once you have identified the correct clone be sure to purify the colony and make a glycerol stock for long term storage It is also a good idea to keep a
7. and the polyhistidine tag 1 Ligate your insert into the appropriate vector and transform into E coli Select transformants on 50 to 100 ug mL ampicillin or 25 50 ug mL Zeocin 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 N terminal peptide 4 Transfect your construct into the cell line of choice using your own method of transfection Generate a stable cell line if desired 5 Test for expression of your recombinant gene by western blot analysis or functional assay For the antibody to the Xpress epitope see page 19 To purify your recombinant protein you may use metal chelating resin such as ProBond ProBond resin is available separately see page 19 for ordering information Methods Cloning into pcDNA 4 HisMax A B and C General Molecular Biology Techniques E coli Host Transformation Method Maintaining pcDNA 4 HisMax 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 We recommend that you propagate vectors co
8. resistant cells should continue to divide at regular intervals to form distinct colonies There should not be any distinct morphological changes in Zeocin resistant cells when compared to cells not under selection with Zeocin To generate a stable cell line expressing your protein you need to determine the minimum concentration of Zeocin required to kill your untransfected host cell line In general concentrations ranging from 50 to 1 000 ng mL Zeocin are sufficient to kill the untransfected host cell line with the average being 250 to 400 ug mL Test a range of concentrations see below to ensure that you determine the minimum concentration necessary for your cell line 1 Seed cells 20 25 confluent for each time point and allow cells to adhere overnight 2 The next day substitute culture medium with medium containing varying concentrations of Zeocin e g 0 50 100 200 400 600 800 and 1 000 pg mL 3 Replenish the selective medium every 3 4 days and observe the percentage of surviving cells 4 Count the number of viable cells at regular intervals to determine the appropriate concentration of Zeocin that prevents growth Select the concentration that kills the majority of the cells in the desired number of days 4 10 days Continued on next page Creation of Stable Cell Lines Continued Possible Sites for Linearization To obtain stable transfectants you may choose to linearize your
9. this resistance we recommend that you place the cells at 4 C for 2 hours after plating be sure to buffer the medium with HEPES Then return the cells to 37 C This stops the cell division process for a short time and allows Zeocin to act Continued on next page 11 Creation of Stable Cell Lines Continued Selecting Stable Integrants Preparing Cells for Lysis Lysis of Cells 12 Once the appropriate Zeocin concentration is determined you can generate a stable cell line with your construct 1 Transfect your cells using the appropriate protocol for your cell line Include a sample of untransfected cells as a negative control 2 After transfection wash the cells once with 1X PBS and add fresh medium to the cells 3 48 hours after transfection split the cells into fresh medium containing Zeocin at the appropriate concentration for your cell line Split the cells such that they are no more than 25 confluent 4 Replenish selective medium every 3 4 days until Zeocin resistant colonies are detected 5 Pick and expand colonies in 96 or 48 well plates Grow cells to near confluence before expanding to larger wells or plates 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 Purification System manual 1 Seed cells in f
10. 4 6 for the sequence of the OBI SP163 element The QBI SP163 element functions as a strong translational enhancer and acts to increase recombinant protein production when placed directly upstream of the ATG initiation codon of the gene of interest The increase in protein expression is thought to occur through ribosome recruitment and a cap independent translation mechanism Stein et al 1998 TM In general expression levels of recombinant protein from pcDNA 4 HisMax are 2 5 fold greater than the levels obtained with the pcDNA 4 His expression vector The amount of recombinant protein expressed will vary depending on the nature of the gene of interest TM The pcDNA 4 HisMax vectors are fusion vectors To ensure proper expression of your recombinant protein you must clone your gene in frame with the ATG at base pairs 1080 1082 This creates a fusion with the N terminal polyhistidine tag Xpress epitope and the enterokinase cleavage site The vector is supplied with the multiple cloning site in three reading frames relative to the N terminal peptide to facilitate cloning See pages 4 6 to develop a cloning strategy If you wish to clone your gene as close as possible to the enterokinase cleavage site follow the guidelines below e Digest pcDNA 4 HisMax A B or C with Kpn I e Create blunt ends with T4 DNA polymerase and dNTPs e Clone your blunt ended insert in frame with the lysine codon AAG of the enterokinase recogn
11. AAACTT AAGCTTAGCG CAGAGGCTTG GGGCAGCCGA QBI SP163 translational enhancer GCGGCAGCCA GGCCCCGGCC CGGGCCTCGG TTCCAGAAGG GAGAGGAGCC CGCCAAGGCG CGCAAGAGAG CGGGCTGCCT CGCAGTCCGA GCCGGAGAGG GAGCGCGAGC CGCGCCGGCC Polyhistidine Region AA CCGGACGGCC TCCGAAACC ATG GGG GGT TCT CAT CAT CAT CAT CAT CAT Met Gly Gly Ser His His His His His His Xpress Epitope HE 2 GGT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC Gly Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp718 1 Kpn BamH BstX I EcoRI Pst 1 A l l GAC GAT GAC GAT AAG GTA CC T AAG GAT CCA GTG TGG TGG AAT TCT GCA vo Asp Asp Asp Lys Val Pro Lys Asp Pro Val Trp Trp Asn Ser Ala Enterokinase recognition site EcoR V GAT Asp CCC Pro TTT Phe ATC Ile GCT Ala GCC Ala EK cleavage site Ban I Not I A O Xba Apa I I I I CAG CAC AGT GGC GGC CGC TCG AGT CTA GAG GGC CCG TTT AAA Gln His Ser Gly Gly Arg Ser Ser Leu Glu Gly Pro Phe Lys BGH reverse priming site Me vn Geen ee aa el GAT CAG CCT CGA CTG TGC CTT CTA GTT GCC AGC CAT CTG TTG Asp Gln Pro Arg Leu Cys Leu Leu Val Ala Ser His Leu Leu CCT CCC CCG TGC CTT CCT TGA CCCTGGAAGG TGCCACTCCC Pro Pro Pro Cys Leu Pro Continued on next page Cloning into pcDNA 4 HisMax A B and C Continued Multiple Cloning Site of Version C Below is the multiple cloning site for ppDNA 4 HisMax C Restriction sites are label
12. DNA stock of your plasmid at 20 C in case you lose the glycerol stock 1 Streak the original colony out on an LB plate containing 50 ug mL ampicillin or 25 ug mL Zeocin in Low Salt LB see page 18 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 25 ug mL Zeocin 3 Grow the culture to mid log phase OD600 0 5 0 7 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 Plasmid Preparation Method of Transfection Positive Control Assay for p galactosidase Activity 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 lipids decreasing transfection efficiency We recommend isolating plasmid DNA using the PureLink HiPure Miniprep Kit or the PureLink HiPure Midiprep Kit see page 19 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 We recommend that you follow exactly the protocol for your cell line Pay particular attention to medium requirements when to pass the cells and at what dilution to split the cells Further information is provided in Current Protocols in Molecular Biology Ausubel et al 1994
13. Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 2009 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 22 invitrogen Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech supporteinvitrogen com For country specific contact information visit our web site at www invitrogen com
14. Reynes J P Stassi D and Tiraby G 1992 A Selectable Bifunctional b Galactosidase Phleomycin resistance Fusion Protein as a Potential Marker for Eukaryotic Cells Gene 114 239 243 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 Calmels T Parriche M Burand H and Tiraby G 1991 High Efficiency Transformation of Tolypocladium geodes Conidiospores to Phleomycin Resistance Curr Genet 20 309 314 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 Drocourt D Calmels T P G Reynes J P Baron M and Tiraby G 1990 Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance Nuc Acids Res 18 4009 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 Goodwin E C and Rottman F M 1992 The 3 Flanking Sequence of the Bovine Growth Hormone Gene Contains Novel Elements R
15. TG ACT GGT GGA CAG Gly Met Ala Ser Met Thr Gly Gly G n Xpress Epitope EE A CAA ATG GGT CGG GAT CTG TAC Gln Met Gly Arg Asp Leu Tyr Asp718 Kpnl BamH A A aha m m GAC GAT GAC GAT AAG GTA CCT AGG Al lC Asp Asp Asp Asp Lys Val Pro Arg I I Enterokinase recognition site EK cleavage site EcoR V BED IF Net l AND le ATA TCC AGC ACA GTG GCG GCC GCT CGA Ile Ser Ser Thr Val Ala Ala Ala Arg BGH reverse priming site BstX EcoRI Pst I CAG TGT GGT GGA ATT CTG CAG Gln Cys Gly Gly Ile Leu Gln me Apa GTC TAG AGGGCCCGTT TAAACCCGCT Val KKK Mi gt re ni GATCAGCCTC GACTGTGCCT TCTAGTTGCC AGCCATCTGT TGTTTGCCCC TCCCCCGTGC Continued on next page Cloning into pcDNA 4 HisMax A B and C Continued Multiple Cloning Site of Version B 821 881 941 1001 1110 1158 1206 1254 1302 1061 Below is the multiple cloning site for pcDNA 4 HisMax B 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 The vector sequence of pcDNA 4 HisMax B is available for downloading from our website www invitrogen com or from Technical Support see page 20 T7 promoter priming site oT CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGC GTTT
16. ability 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 Label License No 5 Invitrogen Technology Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer Limited Use Label License No 77 QBI SP163 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the prod
17. d from pcDNA 4 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 13 14 for more information High level stable and transient expression can be carried out in most mammalian cells The vectors contain the following elements Human cytomegalovirus immediate early CMV promoter for high level expression in a wide range of mammalian cells e QBISP163 translational enhancer for increased levels of recombinant protein expression Stein et al 1998 see page 3 for more information e Three reading frames to facilitate in frame cloning with an N terminal peptide encoding the Xpress epitope and a polyhistidine metal binding tag e Zeocin resistance gene for selection of stable cell lines Mulsant et al 1988 see page 16 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 TM The control plasmid pcDNA 4 HisMax 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 pcDNA 4 HisMax Consult the multiple cloning sites described on pages 4 6 to determine which vector A B or C should be used to clone your gene in frame with the N TM terminal Xpress epitope
18. e 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 2 Scrape cells into 1 mL PBS and pellet the cells at 1 500 x g for 5 minutes 3 Resuspend in 50 uL Cell Lysis Buffer see page 18 Other cell lysis buffers are suitable 4 Incubate cell suspension at 37 C for 10 minutes to lyse the cells Note You may prefer to lyse the cells at room temperature or on ice if degradation of your protein is a potential problem 5 Centrifuge the cell lysate at 10 000 x g for 10 minutes to pellet nuclei and transfer the supernatant to a fresh tube Assay the lysate 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 The N terminal peptide containing the Xpress epitope and the polyhistidine tag will add approximately 3 4 kDa to the size of your protein Note that the QBI SP163 element is not translated You will need 5 x 106 to 1 x 107 transfected cells for purification of your
19. ecipe Note the lower salt content of this medium Failure to use low salt LB medium will result in non selection due to inactivation of the drug Low Salt LB Medium 10 g Tryptone 5 g NaCl 5 g Yeast Extract 1 Combine the dry reagents above and add deionized distilled water to 950 mL Adjust pH to 7 5 with 5 M NaOH Bring the volume up to 1 liter For plates add 15 g L agar before autoclaving Autoclave on liquid cycle at 15 lbs sq in and 121 C for 20 minutes Thaw Zeocin on ice and vortex before removing an aliquot Allow the medium to cool to at least 55 C before adding the Zeocin to 25 ug mL final concentration 5 Store plates at 4 C in the dark Plates containing Zeocin are stable for 1 2 weeks 50 mM Tris HCI pH 7 8 150 mM NaCl 1 Nonidet P 40 1 This solution can be prepared from the following common stock solutions For 100 mL combine 1 M Tris base 5 mL 5 M NaCl 3 mL Nonidet P 40 1 mL 2 Bring the volume up to 90 mL with deionized water and adjust the pH to 7 8 with HCI 3 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 Primers The following products may be used with the pcDNA 4 HisMax vectors For details visit www invitrogen com or contact Technical Support page 20
20. ed to indicate the cleavage site The boxed nucleotide indicates the variable region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pcDNA 4 HisMax C is available for downloading from our website www invitrogen com or from Technical Support see page 20 T7 promoter priming site 821 CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG 7 I 881 GGAGACCCAA GCTGGCTAGC GTTTAAACTT AAGCTTAGCG CAGAGGCTTG GGGCAGCCGA QBI SP163 translational enhancer 941 GCGGCAGCCA GGCCCCGGCC CGGGCCTCGG TTCCAGAAGG GAGAGGAGCC CGCCAAGGCG 1001 CGCAAGAGAG CGGGCTGCCT CGCAGTCCGA GCCGGAGAGG GAGCGCGAGC CGCGCCGGCC Polyhistidine Region 1 1 1061 CCGGACGGCC TCCGAAACC ATG GGG GGT TCT CAT CAT CAT CAT CAT CAT Met Gly Gly Ser His His His His His His Xpress Epitope 1110 GGT ATG GCT AGC ATG ACT GGT GGA CAG CAA ATG GGT CGG GAT CTG TAC Gly Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp718 1 Kpnl BamH BstX EcoR a l mms i 1158 GAC GAT GAC GAT AAG GTA CCA GGA TCC AGT GTG GTG GAA TTC TGC AGA Asp Asp Asp Asp Lys Val Pro Gly Ser Ser Val Val Glu Phe Cys Arg I Enterokinase recognition site EcoR V EK cleavage site en Not Xho Xba Apa I I I 1206 TAT CCA GCA CAG TGG CGG CCG CTC GAG TCT AGA GGG CCC GTT TAA Tyr Pro Ala Gln Trp Arg Pro Leu Glu Ser Arg Gly Pro Val BGH reverse priming site
21. eocin Handling Zeocin Zeocin is used for selection in mammalian cells Mulsant et al 1988 plants Perez et al 1989 yeast Baron et al 1992 and prokaryotes Drocourt et al 1990 Suggested concentrations of Zeocin for selection in mammalian cell lines and E coli are listed below Organism Zeocin Concentration and Selective Medium E coli 25 50 g mL in low salt LB medium see page 18 for recipe Mammalian Cells 50 1000 pg mL varies with cell line Efficient selection requires that the concentration of NaCl be no more than 5 g liter lt 90 mM TM e High salt and acidity or basicity inactivates Zeocin Therefore we recommend that you reduce the salt in bacterial medium and adjust the pH to 7 5 to keep the drug active see page 18 TM e Store Zeocin at 20 C and thaw on ice before use e Zeocin is light sensitive Store drug plates and medium containing drug in the dark e Wear gloves a laboratory coat and safety glasses or goggles when handling solutions containing Zeocin e Zeocin is toxic Do not ingest or inhale solutions containing the drug 17 Recipes Low Salt LB Medium with Zeocin Cell Lysis Buffer 18 For Zeocin to be active the salt concentration of the medium must be low lt 90 mM and the pH must be 7 5 For selection in E coli it is imperative that you prepare LB broth and plates using the following r
22. equired for Efficient and Accurate Polyadenylation J Biol Chem 267 16330 16334 Miller J H 1972 Experiments in Molecular Genetics Cold Spring Harbor New York Cold Spring Harbor Laboratory Mulsant P Tiraby G Kallerhoff J and Perret J 1988 Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells Somat Cell Mol Genet 14 243 252 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 Mol Cell Biol 7 4125 4129 Perez P Tiraby G Kallerhoff J and Perret J 1989 Phleomycin Resistance as a Dominant Selectable Marker for Plant Cell Transformation Plant Mol Biol 13 365 373 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 Stein I Itin A Einat P Skaliter R Grossman Z and Keshet E 1998 Translation of Vascular Endothelial Growth Factor mRNA by Internal Ribosome Entry Implications for Translation under Hypoxia Mol Cell Biol 18 3112 3119 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of
23. er bases 917 1079 ATG initiation codon bases 1080 1082 Polyhistidine tag bases 1092 1109 Xpress epitope bases 1149 1172 Enterokinase recognition site bases 1158 1172 Multiple cloning site bases 1172 1245 BGH reverse priming site bases 1265 1282 BGH polyadenylation sequence bases 1268 1495 f1 origin bases 1541 1969 SV40 promoter and origin bases 1996 2305 EM 7 promoter bases 2353 2408 Zeocin resistance gene bases 2427 2801 SV40 polyadenylation sequence bases 2931 3061 pUC origin bases 3444 4117 Ampicillin resistance gene bases 4262 5122 Continued on next page 13 pcDNA 4 HisMax Vector Continued Features of pcDNA 4 HisMax 14 pcDNA 4 HisMax A 5258 bp pcDNA 4 HisMax B 5259 bp and pcDNA 4 HisMax C 5257 bp contain the following elements All features have been functionally tested Feature Benefit Human cytomegalovirus CMV immediate early promoter enhancer Permits 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 QBI SP163 translational enhancer Increases expression of your recombinant protein via a cap independent translation mechanism Stein et al 1998 N terminal polyhistidine tag Permits purification of your recombinant protein on metal TM
24. gene B lactamase Selection of transformants in E coli pcDNA 4 HisMax lacZ Description pcDNA 4 HisMax lacZ is a 8321 bp control vector containing the gene for B galactosidase This vector was constructed by ligating a 3 1 kb Kpn I EcoR I fragment containing the lacZ gene into the Kpn I EcoR I site of pcDNA 4 HisMax Map of Control The figure below summarizes the features of the pe DNA 4 HisMax lacZ vector Vector The vector sequence for pcDNA 4 HisMax lacZ is available for downloading from our website www invitrogen com or from Technical Support see page 20 SP 163 eco o V x TM nition as o i gt ATG 6xHis Ep one ite i lt 2 la cZ u a pcDNA4 HisMax lacZ Comments for pcDNA4 HisMax lacZ 8321 nucleotides CMV promoter bases 232 819 T7 promoter priming site bases 863 882 QBI SP163 translational enhancer bases 917 1079 ATG initiation codon bases 1080 1082 Polyhistidine tag bases 1092 1109 Xpress epitope bases 1149 1172 Enterokinase recognition site bases 1158 1172 LacZ ORF bases 1197 4247 BGH reverse priming site bases 4328 4345 BGH polyadenylation sequence bases 4331 4558 f1 origin bases 4604 5032 SV40 promoter and origin bases 5059 5368 EM 7 promoter bases 5416 5471 Zeocin resistance gene bases 5490 5864 SV40 polyadenylation sequence bases 5994 6124 pUC origin bases 6507 7180 Ampicillin resistance gene bases 7325 8185 15 Zeocin
25. ifically excludes the right to sell transfer make commercialize or otherwise exploit the QBI SP163 mammalian expression system or its component parts In accepting this license all users acknowledge that QBI Enterprises Ltd makes no warranties express or implied of any kind and hereby disclaims any warranties representations or guarantees of any kind as to the QBI SP163 mammalian expression system product All users intending to use the product other than as licensed above are required to obtain a license from QBI Enterprises Ltd prior to purchase or use for any such other purpose Life Technologies is required by its licensing agreement to report to QBI the identity of all purchasers of the SP163 mammalian expression system Inquiries regarding commercial licenses should be directed to QBI Enterprises Ltd Vice President Business Development Weizmann Scientific Park P O Box 741 Nes Ziona 74106 Israel Tel 972 8 940 8147 Fax 972 8 940 6476 21 References 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 Baron M
26. invitrogen pcDNA 4 HisMax A B and C Catalog no V864 20 Rev Date 31 October 2010 Manual part no 25 0258 MAN0000096 ii Table of Contents Kit Contents and storage nina REN daens lirios iv INTLOGUICH AAA ON 1 POUR a ED 1 Methods coccion 2 Cloning into peDNA 4 HisMax A Band Cana ee 2 Transfection and Analysis sv vassssnsvanstentnens antenne innbruddsraid henne 8 Creation of Stable Cell Lines uterinas 10 O nsii oeae een eenaa one rea eaaa aeaaea aa eaaa aoea aniseed 13 BEDNA 4 HisMaX Vector RER nd 13 PCDNA4 TiSMaxhlacZin renner ME ne 15 LEBEN Bar EN EEE EEE 16 Recipes rar E T gee heide LG sah died cgbteen skal deds seangeasea adda PAGE 18 Accessory Products sannemeike hennes 19 Technical Support anne nalen ine nel a in eb tota 20 P tehaser Netificatl Of tai 21 References van Beed SEE 22 iii Kit Contents and Storage Shipping and Storage Kit Contents pcDNA 4 HisMax vectors are shipped on wet ice Upon receipt store vectors at 20 C All vectors are supplied as detailed below Store the vectors at 20 C Vector Composition Amount pcDNA 4 HisMax A B 40 uL of 0 5 ug pL vector in 10 mM Tris 20 ug and C HCL 1 mM EDTA pH 8 0 pcDNA 4 HisMax lacZ 40 uL of 0 5 ug uL vector in 10 mM Tris 20 ug HCI 1 mM EDTA pH 8 0 Introduction Product Overview Description of the System Experimental Outline pcDNA 4 HisMax A B and C are 5 3 kb vectors derive
27. ition site If you wish to separate your protein of interest from the N terminal peptide tag you may use any suitable enterokinase including EnterokinaseMax from Invitrogen see page 19 Following enterokinase cleavage no vector encoded amino acid residues will be present in your protein Continued on next page Cloning into pcDNA 4 HisMax A B and C Continued Multiple Cloning Site of Version A 821 881 941 1001 1110 1158 1206 1259 1061 Below is the multiple cloning site for pcDNA 4 HisMax A Restriction sites are labeled to indicate the cleavage site The boxed nucleotides indicate the variable region Note that there is a stop codon within the Xba I site The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pcDNA 4 HisMax A is available for downloading from our website www invitrogen com or from Technical Support see page 20 T7 promoter priming site nern CTGGCTAACT AGAGAACCCA CTGCTTACTG GCTTATCGAA ATTAATACGA CTCACTATAG PD GGAGACCCAA GCTGGCTAGC GTTTAAACTT AAGCTTAGCG CAGAGGCTTG GGGCAGCCGA QBI SP163 translational enhancer GCGGCAGCCA GGCCCCGGCC CGGGCCTCGG TICCAGAAGG GAGAGGAGCC CGCCAAGGCG CGCAAGAGAG CGGGCTGCCT CGCAGTCCGA GCCGGAGAGG GAGCGCGAGC CGCGCCGGCC Polyhistidine Region Tg CCGGACGGCC TCCGAAACC ATG GGG GGT TCT CAT CAT CAT CAT CAT CAT Met Gly Gly Ser His His His His His His GGT ATG GCT AGC A
28. ive 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 Centrifuge the cells at 240 x g for 5 minutes Resuspend the cell pellet in PBS Centrifuge the cells at 240 x g for 5 minutes Remove PBS 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 Purification System 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 pcDNA 4 HisMax Vector Map of The figure below summarizes the features of the pcDNA 4 HisMax vectors The pcDNA 4 HisMax sequences for pcDNA 4 HisMax A B and C are available for downloading from our website www invitrogen com or from Technical Support see page 20 Xpress EK Recognition ATG 6xHis Ebitope Site co NG F lt pcDNA4 HisMax A B C There is a stop codon following the Xba I site in version A Comments for pcDNA4 HisMax A 5258 nucleotides CMV promoter bases 232 819 T7 promoter priming site bases 863 882 QBI SP163 translational enhanc
29. ntaining inserts in E coli strains that are recombination deficient recA and endonuclease A deficient end A For your convenience TOP10F is available as chemically competent or electrocompetent cells from Invitrogen see page 19 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 TM To propagate and maintain the peDNA 4 HisMax vectors use a small amount of the supplied 0 5 ug puL stock solution in TE pH 8 0 to transform a recA end A E coli strain like TOP10F DH5a JM109 or equivalent Select transformants on LB plates containing 50 to 100 ug mL ampicillin or 25 to 50 pg mL Zeocin in Low Salt LB Be sure to prepare a glycerol stock of each plasmid for long term storage see page 7 for protocol Continued on next page Cloning into pcDNA 4 HisMax A B and C Continued QBI SP163 Translational Enhancer Note The QBI SP163 element is a 163 nucleotide splice variant derived from the 5 untranslated region UTR of the mouse vascular endothelial growth factor VEGF gene Stein et al 1998 The splice variant is composed of a 31 nucleotide fragment containing the 5 cap sequence of the VEGF gene fused to a 132 nucleotide fragment of the 5 UTR immediately preceding the translational start site of the VEGF gene Refer to the diagrams on pages
30. nts owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for research use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or e mail outlicensing lifetech com 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 The QBI SP163 mammalian expression system technology is owned by QBI Enterprises Ltd and licensed to Life Technologies Purchasers of this product are granted a license to use the QBI SP163 mammalian expression system only for internal research purposes which license spec
31. protein on a 2 mL ProBond column or other metal chelating column Refer to the manufacturer s instructions before attempting to purify your fusion protein To prepare cells for lysis refer to the protocol on page 12 Creation of Stable Cell Lines Introduction Effect of Zeocin on Sensitive and Resistant Cells Selection in Mammalian Cell Lines 10 pcDNA 4 HisMax vectors contain the Zeocin resistance gene for selection of stable cell lines using Zeocin We recommend that you test the sensitivity of your mammalian host cell to Zeocin as natural resistance varies among cell lines General information and guidelines are provided below for your TM convenience For more information about Zeocin refer to page 16 The method of killing with Zeocin is quite different from neomycin and hygromycin Cells do not round up and detach from the plate Sensitive cells TM may exhibit the following morphological changes upon exposure to Zeocin e Vast increase in size similar to the effects of cytomegalovirus infecting permissive cells e Abnormal cell shape e Presence of large empty vesicles in the cytoplasm breakdown of the endoplasmic reticulum and golgi apparatus or other scaffolding proteins e Breakdown of plasma and nuclear membrane appearance of many holes in these membranes e Eventually these cells will completely break down and only cellular debris will remain Zeocin
32. uct in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of pate
33. vector before 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 unique sites that may be used to linearize your construct prior to transfection Other restriction sites are possible Note that for the enzymes listed below the cleavage site is indicated for versions A B and C of pcDNA 4 HisMax Be sure that your insert does not contain the restriction enzyme site you wish to use to linearize your vector Enzyme Restriction Site bp Location Supplier A B C Bgl II 12 Upstream of CMV promoter Many Mfe I 161 Upstream of CMV promoter New England Biolabs Nru I 208 Upstream of CMV promoter Many Bst1107 I 3063 A 3064 B 3062 C End of SV40 poly A AGS Fermentas Takara Boehringer Mannhiem Eam1105 I 4335 A 4336 B 4334 C Ampicillin gene AGS Fermentas Takara Fsp I 4557 A 4558 B 4556 C Ampicillin gene Many Pvul 4705 A 4706 B 4704 C Ampicillin gene Many Sca I 4815 A 4816 B 4814 C Ampicillin gene Many Ssp I 5139 A 5140 B 5138 C Backbone Many Angewandte Gentechnologie Systeme Selection Tip TM Some cells may be more resistant to Zeocin than others If cells are dividing TM rapidly Zeocin may not be effective at low concentrations To overcome
Download Pdf Manuals
Related Search