pcDNA4/Myc-His A, B and C - Thermo Fisher Scientific
Contents
1. 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 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 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 Nucleic Acids Res 18 4009 Evans G L 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 32 115 121 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 Required for Efficient and Accurate Polyadenylation J Biol Chem 267 16330 16334 Kozak M 1987 An Analysis of 5 Noncoding Sequences from 699 Vertebrate Messenger RNAs Nucleic Acids Res 15 8125 81482. Kozak M 1991 An Analysis of Vertebrate mRNA Sequences Intimations of Translational Control J Cell Biology 115 887 903 Kozak M 1990 Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 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 Continued on next page 21 References Continued 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 Molec 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 Harb
3. There is a unique BstE Il site but no Xba lor Apa sites in version C There is a unique Sac Il site between the Apa site and the BstB site in version B only Comments for pcDNA4 Myc His 5075 nucleotides CMV promoter bases 209 863 T7 promoter priming site bases 863 882 Multiple cloning site bases 902 999 myc epitope bases 997 1026 Polyhistidine tag bases 1042 1059 BGH reverse priming site bases 1082 1099 BGH polyadenylation signal bases 1085 1312 f1 origin bases 1358 1786 SV40 promoter and origin bases 1814 2122 EM 7 promoter bases 2170 2225 Zeocin resistance gene bases 2244 2618 SV40 polyadenylation signal bases 2748 2878 pUC origin bases 3261 3934 Ampicillin resistance gene bases 4079 4939 Continued on next page 11 pcDNA 4 myc His Vector Continued Features of pcDNA 4 myc His A 5075 bp pcDNA 4 myc His B 5079 bp and pcDNA 4 myc His pcDNA 4 myc His C 5071 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 Multiple cloning site in three reading frames Allows insertion of your gene and fa
4. and endonuclease A deficient endA For your convenience TOP10F is available from Invitrogen as chemically competent or electrocompetent cells 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 TM To propagate and maintain the pcDNA 4 myc 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 to 100 ug mL ampicillin or 25 to 50 ug mL Zeocin in Low Salt LB Be sure to prepare a glycerol stock of each plasmid for long term storage see page 5 Your insert should contain a Kozak translation initiation sequence for proper initiation of translation Kozak 1987 Kozak 1991 Kozak 1990 An example of a Kozak consensus sequence is provided below Note that other sequences are possible but the A at position 3 and the G at position 4 are the most critical for function shown in bold The ATG initiation codon is shown underlined ANNATGG To express your gene as a recombinant fusion protein you must clone your gene in frame with the C terminal peptide The vector is supplied in three reading frames to facilitate cloning See pages 3 4 to develop a cloning strategy If you wish to express your p
5. see below to ensure that you determine the minimum concentration necessary for your cell line 1 Seed cells 2 x 10 cells 60 mm plate 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 50 125 250 500 750 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 TM appropriate concentration of Zeocin that prevents growth Continued on next page Creating Stable Cell Lines Continued Possible Sites for Linearization To obtain stable transfectants you may choose to linearize your 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 transformation Other restriction sites are possible Note that the cleavage site is indicated for versions A B and C of pcDNA 4 myc His 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 Bel II 13 Upstream of CMV promoter Many
6. Mfe I 161 Upstream of CMV promoter New England Biolabs Nru I 209 Upstream of CMV promoter Many Mlu I 229 5 end of CMV promoter Many Bst1107 I 2881 A 2885 B 2877 C End of SV40 poly A AGS Fermentas Takara Boehringer Mannhiem Eam1105 I 4153 A 4157 B 4149 C Ampicillin gene AGS Fermentas Takara Fsp I 4375 A 4379 B 4371 C Ampicillin gene Many Pvu I 4523 A 4527 B 4519 C Ampicillin gene Many Sca I 4633 A 4637 B 4629 C Ampicillin gene Many Ssp I 4957 A 4961 B 4953 C Ampicillin gene Many Angewandte Gentechnologie Systeme Selecting Stable Integrants stable cell line with your construct Once the appropriate Zeocin concentration is determined you can generate a 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 Continued on next page Creating Stable Cell Lines Continued Preparing Cells for Use the procedure below to prepare cells for lysis pr
7. O NH Continued on next page 14 Zeocin Continued Applications of Zeocin 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 ug mL in low salt LB medium see page 16 for recipe Mammalian Cells 50 1 000 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 16 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 15 Recipes Low Salt LB Medium with Zeocin Cell Lysis Buffer 16 For Zeocin to be active the salt concentration of the medium must remain low lt 90 mM and the pH must be 7 5 For
8. the Lipofectamine 2000 Reagent for mammalian transfection For more details call Technical Support see page 19 or visit our website at www invitrogen com TM pcDNA 4 myc His lacZ is provided as a positive control vector for mammalian cell transfection and expression see page 13 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 P 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 Continued on next page Transfection and Analysis Continued Detecting Fusion Proteins Note Purification Several antibodies are available from Invitrogen to detect expression of your fusion protein from pcDNA 4 myc His see page 18 To detect 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 2 Scrap
9. Note that there are two BstX I sites in the polylinker TM Multiple Cloning Below is the multiple cloning site for p DNA 4 myc His B Restriction sites are Site of Version B 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 myc His B is available for downloading from our website www invitrogen com or from Technical Support see page 19 T7 promoter priming site Pina lll Acc65 Ann Banin l 861 ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGT TAAG CIT cer ACC GAG CTC GGA Leu Gly Thr Glu Leu Gly Balk I EcoR Est ECORV Br I Noti 923 TCC ACT AGT CCA GTG TGG TGG AAT TCT GCA GAT ATC CAG CAC AGT GGC GGC CGC Ser Thr Ser Pro Val Trp Trp Asn Ser Ala Asp Ile Gln His Ser Gly Gly Arg Xho l Abe l Ap l 380 II BB l myc epitope 977 TCG AUT CTA GAG GGC CCG CGG TTC GAA CAA AAA CTC ATC TCA GAA GAG GAT Ser Ser Leu Glu Gly Pro Arg Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Agel Polyhistidine tag Pme ed l 1028 CTG AAT ATG CAT ACC GGT CAT CAT CAC CAT CAC CAT TGA GTTT AAACCCGCTG Leu Asn Met His Thr Gly His His His His His His BGH Reverse priming site 1 1081 ATCAGCCTCG ACTGTGCCTT CTAGTTGCCA Note that there are two BstX I sites in the polylinker Continued on next page Cloning into pcDNA 4 myc His A B and C Continued Multiple Cloning Si
10. R952 25 Anti His C term Detects the C terminal polyhistidine R930 25 Anti His C term HRP 8 requires the free carboxyl group R931 25 for detection Lindner et al 1997 Anti His C term AP R932 25 HHHHHH COOH Technical Support Web Resources Visit the Invitrogen website at www invitrogen com for e Technical resources including manuals vector maps and sequences application notes 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 Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail tech supportQinvitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Certificate of Analysis Limited Warranty Material Safety Data Sheets MSDSs are available on our website at www invitrogen com msds The Certificate
11. ampicillin or 25 50 g mL Zeocin in Low Salt LB see page 16 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 DH5aF IO SURE SURE2 encodes the ble bleomycin resistance gene These strains will confer resistance to Zeocin For the most efficient selection we recommend that you choose an E coli strain that does not contain the Tn5 gene Le TOP10 DH5a DH10 etc Continued on next page Cloning into pcDNA 4 myc His A B and C Continued I We recommend that you sequence your construct with the T7 Forward and BGH NS nr Reverse primers to confirm that your gene is fused in frame with the myc epitope Ta E and the C terminal polyhistidine tag For ordering primers see page 17 Preparing a Once you have identified the correct clone be sure to purify the colony and make Glycerol Stock a 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 on an LB plate containing 50 ug mL ampicillin or 25 ug mL Zeocin in Low Salt LB Incubate the plate at 37 C overnight 2 Isolate a single colony and inoculate into 1 2 mL of LB with 50 ng mL ampicillin 3 Grow the culture to mid log phase ODgoo 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
12. and Analysis Introduction Plasmid Preparation Methods of Transfection Positive Control Assay for p galactosidase Activity Once you have confirmed that your construct is in the correct orientation and fused in frame with the C terminal peptide 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 lipids decreasing transfection efficiency We recommend isolating plasmid DNA using the PureLink HiPure Miniprep Kit 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 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 see page 21 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
13. anscription termination and polyadenylation of mRNA pUC origin High copy number replication and growth in E coli Ampicillin resistance gene P lactamase Selection of transformants in E coli 12 pcDNA 4 myc His lacZ Map of Control Vector Comments for pcDNA4 Myc His lacZ 8120 nucleotides CMV promoter bases 209 863 T7 promoter priming site bases 863 882 pcDNA 4 myc His lacZ is a 8120 bp control vector containing the gene for B galactosidase This vector was constructed by ligating a 3 880 bp BamH I Stu I fragment containing the CMV promoter and the Zeocin resistance gene from pcDNA 4 myc His B to a 4 240 bp BamH I Stu I fragment containing the lacZ gene myc epitope and polyhistidine tag from pcDNA 3 1 myc His lacZ The figure below summarizes the features of the peDNA 4 myc His lacZ vector The vector sequence for pcCDNA 4 myc His lacZ is available for downloading from our website www invitrogen com or by contacting Technical Support see page 19 pcDNA4 myc His lacZ 8 1 kb LacZ ORF bases 963 4019 myc epitope bases 4044 4073 Polyhistidine tag bases 4089 4106 BGH reverse priming site bases 4129 4146 BGH polyadenylation signal bases 4132 4359 f1 origin bases 4405 4833 SV40 promoter and origin bases 4861 5169 EM 7 promoter bases 5217 5272 Zeocin resistance gene bases 5291 5665 SV40 polyadenylation signal bases 5795 5925 pUC origin bases 6308 6981 Ampi
14. cilitates cloning in frame with the myc epitope and polyhistidine C terminal tag myc epitope Glu Gln Lys Leu Ie Ser Glu Glu Asp Leu Allows detection of your recombinant protein with the Anti myc Antibody the Anti myc HRP Antibody or the Anti myc AP Antibody Evans et al 1985 see page 18 for ordering C terminal polyhistidine 6xHis tag Permits purification of your recombinant protein on metal chelating resin such as ProBond In addition the C terminal polyhistidine tag is the epitope for the Anti His C term Antibody the Anti His C term HRP Antibody and the Anti His C term AP Lindner et al 1997 see page 18 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 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 tr
15. cillin resistance gene bases 7126 7986 13 Zeocin TM Introduction The pcDNA 4 myc His 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 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 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 in a stoichiometric manner to inhibit its DNA strand cleavage activity Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin Molecular Weight The formula for Zeocin is CssHscOz1N2092Cu HCI and the molecular weight is Formula and 1 527 5 daltons Zeocin is an HCI salt The diagram below shows the structure Structure of Zeocin H CONH2 O H2 N N R e u 9 N N s NH N AN vo Cua T CH s ET H N a
16. e 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 16 Other lysis buffers may be suitable 4 Incubate cell suspension at 37 C for 10 minutes to lyse the cells 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 C terminal peptide containing the myc epitope and the polyhistidine tag will add approximately 3 kDa to the size of your protein You will need 5 x 10 to 1 x 107 transfected cells for purification of your 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 10 Creating Stable Cell Lines Introduction Effect of Zeocin on Sensitive and Resistant Cells Selection in Mammalian Cell Lines The pcDNA 4 myc His vectors contain the Zeocin resistance gene for selection of stable cell l
17. e information see page 16 3 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 using your own method of transfection Generate a stable cell line if desired 6 Test for expression of your recombinant gene by western blot analysis or functional assay For antibodies to the myc epitope or the C terminal polyhistidine tag see page 18 7 To purify your recombinant protein you may use metal chelating resin such as ProBond ProBond resin is available separately see page 17 Methods Cloning into pcDNA 4 myc His A B and C General Molecular Biology Techniques E coli Strain Transformation Method Maintaining pcDNA 4 myc 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 growth of this vector including TOP10F DH5aF JM109 and INVaF We recommend that you propagate vectors containing inserts in E coli strains that are recombination deficient recA
18. en com or contact Technical Support page 19 Item Amount Catalog no 6 x 2 mL precharged prepacked K850 01 ProBond Purification ProBond resin columns and System buffers for native and denaturing purification A 50 mL R801 01 ProBond Resin 150 mL R801 15 Electrocomp TOP10F 5 x 80 uL C665 55 One Shot TOP10F 21 x 50 uL C3030 03 chemically competent cells EKMax Enterokinase 250 units E180 01 PureLink HiPure Plasmid Miniprep Kit 100 preps K2100 03 PureLink HiPure Plasmid Midiprep Kit 25 preps K2100 04 P Gal Assay Kit 80 mL K1455 01 P Gal Staining Kit 1 kit K1465 01 sl 1 gram R250 01 Zeocin 5 grams R250 05 Lipofectamine 2000 Reagent 0 75 mL 11668 027 Primers For your convenience Invitrogen offers a custom primer synthesis service Visit www invitrogen com for more details Continued on next page 17 Accessory Products Continued Antibodies 18 If you do not have an antibody specific to your protein Invitrogen offers the Anti myc 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 myc Detects a 10 amino acid epitope R950 25 Anti myc HRP derived from c myc Evan et al 1985 R951 25 Anti myc AP KONSERN
19. ines 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 TM your convenience For more information about Zeocin refer to page 14 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 will TM exhibit the following morphological changes upon exposure to Zeocin e Vast increase in size e Abnormal cell shape e Presence of large empty vesicles in the cytoplasm breakdown of the endoplasmic reticulum and golgi apparatus or scaffolding proteins e Breakdown of plasma and nuclear membrane appearance of many holes in these membranes Eventually these cells will completely break down and only strings of protein will remain Zeocin 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 Typically concentrations between 50 and 1 000 ng mL Zeocin are sufficient to kill the untransfected host cell line Test a range of concentrations
20. invitrogen pcDNA 4 myc His A B and C Catalog no V863 20 Rev Date 27 October 2010 Manual part no 25 0236 MAN0000078 ii Table of Contents Kit Contents nd Storage sun nennen deed send iv Introduction ae 1 Product ONE Wi isa 1 MethodsS A eneen edad 2 Cloning into ppDNA 4 myc His A B and Ei silanen ans 2 Transtection and AN alysis odian lt ii pi 6 Creating Stable Cell Lines an danseres taaan ava e eaea caera dolencia das benee beard 8 APpendix NM arad aa are aa aaan ii Saidatina 11 PEDNA 4s myc His Veelers ennn A 11 AN a EY AAA A as EE REN R a Eaa a EN ERE 13 ZOOCH ts EE GE 14 Recipes 2 222 ae 16 Accessory Products A eee Ab 17 Technieal Support PP ensen an Herentalse 19 P rch ser N tifie tion 22 2s2 228er E E dna 20 References sir 21 iii Kit Contents and Storage Shipping and Storage Kit Contents at 20 C pcDNA 4 myc His vectors are shipped on wet ice Upon receipt store vectors All vectors are supplied as detailed below Store the vectors at 20 C Item Composition Amount pcDNA 4 myc His A B and C 40 uL of 0 5 ug uL vector in 10 mM Tris HCl 20 ug 1 mM EDTA pH 8 0 40 uL of 0 5 ug uL vector in 10 mM Tris HCI 20 ug pcDNA 4 myc His lacZ 1 mM EDTA pH 8 0 Introduction Product Overview Description of the System Experimental Outline TM pcDNA 4 myc His A B and C are 5 1 kb vectors designed for overproducti
21. ior to purification of your Lysis Lysis of Cells 10 TM protein on ProBond You will need 5 x 10 to 1 x 107 cells for purification of TM TM your protein on a 2 mL ProBond column see ProBond Purification System manual 1 Seed cells in five 1 75 flasks or 2 to 3 T 175 flasks 2 Grow the cells in selective medium until they are 80 90 confluent 3 Harvest the cells by treating with trypsin EDTA for 2 to 5 minutes or by scraping the cells in PBS 4 Inactivate the trypsin by diluting with fresh medium if necessary and transfer the cells to a sterile microcentrifuge tube 5 Centrifuge the cells at 240 x g for 5 minutes Resuspend the cell pellet in PBS 6 Centrifuge the cells at 240 x g for 5 minutes You may lyse the cells immediately or freeze in liquid nitrogen and store at 80 C until needed 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 myc His Vector Map of The figure below summarizes the features of the pcDNA 4 myc His vectors pcDNA 4 myc His The vector sequences for ppDNA 4 myc His A B and C are available for downloading from our website www invitrogen com or from Technical Support see page 19 pcDNA4 myc His A B C 5 1 kb
22. 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 19 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer 20 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its com ponents 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
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24. on of recombinant proteins in mammalian cell lines Features of the vectors allow purification and detection of expressed proteins see pages 11 12 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 Three reading frames to facilitate in frame cloning with a C terminal peptide encoding the myc c myc epitope and a polyhistidine 6xHis metal binding tag e Zeocin resistance gene for selection of stable cell lines Mulsant et al 1988 see page 14 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 COS7 TM The control plasmid pcDNA 4 myc 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 pcDNA 4 myc His 1 Consult the multiple cloning sites described on pages 3 4 to determine which vector A B or C to use for cloning your gene in frame with the C terminal myc epitope and the polyhistidine tag 2 Ligate your insert into the appropriate vector and transform into E coli Select transformants on 50 to 100 pg mL ampicillin or 25 to 50 ug mL Zeocin in Low Salt LB For mor
25. or 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 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 2009 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_support invitrogen com For country specific contact information visit our web site at www invitrogen com
26. rotein WITHOUT the C terminal peptide be sure to include a stop codon Continued on next page Cloning into pcDNA 4 myc His A B and C Continued TM Multiple Cloning Below is the multiple cloning site for p DNA 4 myc His A Restriction sites are Site of Version A 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 of pcDNA 4 myc His A is available for downloading from our website www invitrogen com or from Technical Support see page 19 T7 promoter priming site Hind II Acc65 1 Kpn BamH 861 ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGT TAA GCT TGG TAC CGA GCT CGG Ala Trp Tyr Arg Ala Arg BstX EcoRI ESI l EGOR V us Not 922 ATC CAC TAG TCC AGT GTG GTG GAA TTC TGC AGA TAT CCA GCA CAG TGG CGG CCG Ile His Ser Ser Val Val Glu Phe Cys Arg Tyr Pro Ala Gln Trp Arg Pro Xho I Xba I Apa I BstB myc epitope l 976 CTC GAG ECT AGA GGG cc TTC GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT Leu Glu Ser Arg Gly Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Agel Polyhistidine tag Pme FP 1030 ATG CAT Acc GGT CAT CAT CAC CAT CAC CAT TGA GTTTAAACCC GCTGATCAGC Met His Thr Glu His His His His His His BGH Reverse priming site 1 1083 CTCGACTGTG CCTTCTAG
27. selection in E coli it is imperative that you prepare LB broth and plates using the following recipe 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 2 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 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 5mL 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 TM Introduction The following products may be used with the pcDNA 4 myc His vectors For details visit www invitrog
28. 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 patents 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 produc
29. t 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 References Andersson S Davis D L Dahlback 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 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
30. te of Version C TM Below is the multiple cloning site for pcDNA 4 myc His C Restriction sites are labeled to indicate the cleavage site The boxed nucleotides indicate the variable region The multiple cloning site has been confirmed by sequencing and functional testing The vector sequence of pcDNA 4 myc His C is available for downloading from our website www invitrogen com or from Technical Support see page 19 T7 promoter priming site Hed III Ar l Kpn l l l 861 ATTAATACGA CTCACTATAG GGAGACCCAA GCTGGCTAGT TA AGC TTG GTA CCG AGC ai 918 TCG GAT Ser Asp Neel 969 GCG GCC Ala Ala A 1020 CTG AAT Leu Asn Ser Leu Val Pro Ser BSIK FOR Pst l EGOR V BSI CCA CTA GTC CAG TGT GGT GGA ATT CTG CAG ATA TCC AGC ACA GIG Pro Leu Val Gln Cys Gly Gly Ile Leu Gln Ile Ser Ser Thr Val Xho l BstE II BstB myc epitope GCT CGA GGT CAC CCA TTC GAA CAA AAA CTC ATC TCA GAA GAG GAT Ala Arg Gly His Pro Phe Glu Gln Lys Leu Ile Ser Glu Glu Asp Age Polyhistidine tag Pme 1 ATG CAT ACC GGT CAT CAT CAC CAT CAC CAT TGA GTTTAAACCC Met His Thr Gly His His His His His His BGH Reverse priming site l 1069 GCTGATCAGC CTCGACTGTG CCTTCTAGTT GC Note that there are two BstX I sites in the polylinker E coli Transformation Important 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
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