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pcDNA™3.1(+) - Thermo Fisher Scientific

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1. 3 Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents growth within 1 3 weeks after addition of the antibiotic Once you have determined the appropriate Geneticin concentration to use for selection you can generate a stable cell line expressing your peDNA 3 1 construct Geneticin is available separately from Invitrogen see page vi for ordering information Use as follows Prepare Geneticin in a buffered solution e g 100 mM HEPES pH 7 3 2 Use the predetermined concentration of Geneticin in complete medium 3 Calculate concentration based on the amount of active drug 4 Cells will divide once or twice in the presence of lethal doses of Geneticin so the effects of the drug take several days to become apparent Complete selection can take from 2 to 3 weeks of growth in selective medium continued on next page Creating Stable Cell Lines continued Possible Sites for Linearizatio of pcDNA3 1 Prior to transfection we recommend that you linearize the peDNA 3 1 vector n Linearizing pcDNA 3 1 will decrease the likelihood of the vector integrating into the genome in a way that disrupts the gene of interest or other elements required for expression in mammalian cells The table below lists unique restriction sites that may be used to linearize your construct prior to transfection Other unique restriction sites are poss
2. purification of single stranded DNA DNA sequencing and DNA biochemistry please 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 DH5 T18 and TOP10 We recommend that you propagate vectors containing inserts in E coli strains that are recombination deficient recA and endonuclease A deficient endA TM If you wish to express a human gene of interest from pcDNA 3 1 we recommend using an Ultimate Human ORF hORF Clone available from Invitrogen For more information about the Ultimate hORF Clones available refer to our Web site www invitrogen com or contact Technical Support page 13 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 pcDNA 3 1 use 10 ng of vector to transform a recA endA E coli strain like TOP10F DH5 T1 TOP10 or equivalent Select transformants on LB plates containing 50 100 pg ml ampicillin Be sure to prepare a glycerol stock of your plasmid containing E coli strain for long term storage see page 5 pcDNA 3 1 and pcDNA 3 1 are non fusion vectors Your insert should contain a Kozak translation ini
3. 4 Hama Cho Nihonbashi Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 3663 7972 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 3663 8242 Tech Fax 44 0 141 814 6117 E mail tech_support invitrogen com E mail jpinfo invitrogen com E mail eurotech invitrogen com MSDS Material Safety Data Sheets MSDSs are available on our website at Certificate of Analysis Limited Warranty www invitrogen com msds The Certificate of Analysis CofA provides detailed quality control information for each product CofAs are available on our website at www invitrogen com support and are searchable by product lot number which is printed on each box Invitrogen is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives Invitrogen warrants that all of its products will perform according to specifications stated on the certificate of analysis The company will replace free of charge any product that does not meet those specifications This warranty limits Invitrogen Corporation s liability only to the cost of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with in
4. COS 7 TM The control plasmid peDNA 3 1 CAT is included for use as a positive control for transfection and expression in the cell line of choice TM Use the following outline to clone and express your gene of interest in pcDNA 3 1 1 Consult the multiple cloning sites described on pages 3 4 to design a strategy to clone your gene into pcDNA 3 1 2 Ligate your insert into the appropriate vector and transform into E coli Select transformants on LB plates containing 50 100 pg ml ampicillin 3 Analyze your transformants for the presence of insert by restriction digestion Select a transformant with the correct restriction pattern and use sequencing to confirm that your gene is cloned in the proper orientation 5 Transfect your construct into the mammalian cell line of interest using your own method of choice Generate a stable cell line if desired 6 Test for expression of your recombinant gene by western blot analysis or functional assay Cloning into pcDNATM3 1 Introduction General Molecular Biology Techniques E coli Strain Note Transformation Method Maintenance of pcDNA 3 1 Cloning Considerations Diagrams are provided on pages 3 4 to help you design a cloning strategy for ligating your gene of interest into peDNA 3 1 General considerations for cloning and transformation are listed below For help with DNA ligations E coli transformations restriction enzyme analysis
5. P 1987 Electroporation for the Efficient Transfection of Mammalian Cells with DNA Nuc Acids Res 15 1311 1326 Felgner P L Holm M and Chan H 1989 Cationic Liposome Mediated Transfection Proc West Pharmacol Soc 32 115 121 Felgner P L and Ringold G M 1989 Cationic Liposome Mediated Transfection Nature 337 387 388 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 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 Eukaryotic Ribosomes Proc Natl Acad Sci USA 87 8301 8305 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 Neumann J R Morency C A and Russian K O 1987 A Novel Rapid Assay for Chloramphenicol Acetyltransferase Gene Expression BioTechniques 5 444 447 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Sec
6. multiple cloning sites are shown on page 3 for pcDNA 3 1 and page 4 for pcDNA 3 1 pcDNA3 1 5428 5427 bp Comments for pcDNA3 1 5428 nucleotides CMV promoter bases 232 819 T7 promoter priming site bases 863 882 Multiple cloning site bases 895 1010 pcDNA3 1 BGH reverse priming site bases 1022 1039 BGH polyadenylation sequence bases 1028 1252 f1 origin bases 1298 1726 SV40 early promoter and origin bases 1731 2074 Neomycin resistance gene ORF bases 2136 2930 SV40 early polyadenylation signal bases 3104 3234 pUC origin bases 3617 4287 complementary strand Ampicillin resistance gene bla bases 4432 5428 complementary strand ORF bases 4432 5292 complementary strand Ribosome binding site bases 5300 5304 complementary strand bla promoter P3 bases 5327 5333 complementary strand PUC ori continued on next page pcDNA 3 1 Vectors continued Features pcDNA 3 1 5428 bp and pcDNA 3 1 5427 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 forward or rever
7. nos N560 02 and N575 02 respectively to confirm that your gene is in the correct orientation for expression and contains an ATG and a stop codon Please refer to the diagrams on pages 3 4 for the sequences and location of the priming sites The primers are available separately from Invitrogen in 2 ug aliquots Once you have identified the correct clone purify the colony and make a glycerol stock for long term storage You should keep a DNA stock of your plasmid at 20 C e Streak the original colony out on an LB plate containing 50 ug ml ampicillin Incubate the plate at 37 C overnight e Isolate a single colony and inoculate into 1 2 ml of LB containing 50 pg ml ampicillin e Grow the culture to mid log phase ODgoo 0 5 0 7 e Mix 0 85 ml of culture with 0 15 ml of sterile glycerol and transfer to a cryovial Store at 80 C continued on next page Transfection Introduction Plasmid Preparation Methods of Transfection Positive Control Assay for CAT Protein Once you have verified that your gene is cloned in the correct orientation and contains an initiation ATG and a stop codon you are ready to transfect your cell line of choice We recommend that you include the positive control vector and a mock transfection negative control to evaluate your results Once you have generated your expression clone you must isolate plasmid DNA for transfection Plasmid DNA for transfection into eukaryotic
8. pages 10 11 enhancer region 3 end 689 CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAAATGTCG 749 TAACAACTCC GCCCCATTGA 3 end of hCMV 809 AAGCAGAGCT CTCTGGCTAA T7 promoter primer bind ing site 869 GACTCACTAT AGGGAGACCC Bs l l TGCTGGATAT 929 GCCGCCACTG Asp718 1 KpnI Hind II stXI EcoRV Afi il Pmel I Mh tte tat 989 GGTACCAAGC TTAAGTTTAA 1049 ATCTGTTGTT TGCCCCTCCC BGH poly A site 1109 CCTTTCCTAA TAAAATGAGG CAAT Bez TATA en CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT putative transcriptional start m CTAGAGAACC CACTGCTTAC TGGCTTATCG AAATTAATAC Nhe I l AAGCTGGCT A EcoRI I CTGCAGAAT ACCGCTGAT CCGTGCCTT AAATTGCAT uy Pme 1 Apa XbaI Xho 1 Not l I m I l GCGTTTAAAC GGGCCCTCTA GACTCGAGCG BsiX I bare I CCACCACACT GGACTAGTGG ATCCGAGCTC peDNA3 1 BGH reverse priming site 1 T AGCCTCGACT GTGCCTTCTA GTTGCCAGCC CTTGACCCTG GAAGGTGCCA CTCCCACTGT Please note that there are two BstX I sites in the polylinker Cloning into pcDNATM3 1 continued E coli Transformation Preparing a Glycerol Stock Once you have obtained a clone containing your gene of interest you may transform the clone into a suitable E coli host see below We recommend including a negative control in your experiment to help you evaluate your results We recommend that you sequence your construct with the T7 Promoter and BGH Reverse primers Catalog
9. vector for mammalian transfection and expression see page 12 and may be used to optimize transfection conditions for your cell line The gene encoding chloramphenicol acetyl transferase CAT is expressed in mammalian cells under the control of the CMV promoter A successful transfection will result in CAT expression that can be easily assayed see below You may assay for CAT expression by ELISA assay western blot analysis fluorometric assay or radioactive assay Ausubel et al 1994 Neumann et al 1987 If you wish to detect CAT protein using western blot analysis you may use the Anti CAT Antiserum Catalog no R902 25 available from Invitrogen Other kits to assay for CAT protein using ELISA assay are available from Roche Molecular Biochemicals Catalog no 1 363 727 and Molecular Probes Catalog no F 2900 continued on next page Creating Stable Cell Lines Introduction Z G SS I c gE Or gt Non I Geneticin Determining Antibiotic Sensitivity Geneticin Selection Guidelines The pcDNA 3 1 and pcDNA 3 1 vectors contain the neomycin resistance gene for selection of stable cell lines using neomycin Geneticin We recommend that you test the sensitivity of your mammalian host cell to Geneticin as natural resistance varies among cell lines General information and guidelines are provided in this section for your convenience TM To obtain stable transfectants we reco
10. ATGTCG CAAT HA TATA nr TAACAACTCC GCCCCATTGA CGCAAATGGG CGGTAGGCGT GTACGGTGGG AGGTCTATAT putative transcriptional start AAGCAGAGCT CTCTGGCTAA CTAGAGAACC CACTGCTTAC 1 3 end ofhCMV T7 promoter primer binding site dd dey GACTCACTAT AGGGAGACCC AAGCTGGCTA GGATCCACTA GTCCAGTGTG Xba I Apa I Pmel AGTCTAGAGG GCCCGTTTAA CATCTGTTGT TTGCCCCTCC BGH poly A site TCCTTTCCTA ATAAAATGAG Nhe 1 BstX I EcoRI GTGGAATTCT ACCCGCTGAT CCCGTGCCTT GAAATTGCAT Pmel Aflil l GCGTTTAAAC 1 EcoR V Hind II Asp7181 KpnI I TGGCTTATCG AAATTAATAC TTAAGCTTGG TACCGAGCTC Xho I I GCAGATATCC AGCACAGTGG CGGCCGCTCG pcDNA3 1 BG H reverse priming site 1 CAGCCTCGAC 1 TGTGCCTTCT AGTTGCCAGC CCTTGACCCT GGAAGGTGCC ACTCCCACTG Please note that there are two BstX I sites in the polylinker continued on next page Cloning into pcDNATM3 1 continued Multiple Cloning Below is the multiple cloning site for p DNA 3 1 gt Restriction sites are labeled to Site of indicate the cleavage site The Xba I site contains an internal stop codon pcDNA 3 1 TCTAGA The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pcDNA 3 1 is available for downloading from our web site www invitrogen com or from Technical Support see page 13 For a map and a description of the features of pcDNA 3 1 please see the Appendix
11. Sca I 4984 Ampicillin gene Invitrogen Catalog no 15436 017 Ssp I 5308 bla promoter Invitrogen Catalog no 15458 011 Angewandte Gentechnologie Systeme continued on next page Creating Stable Cell Lines continued Selection of Once you have determined the appropriate Geneticin concentration to use for selection in your Stable host cell line you can generate a stable cell line expressing your gene of interest Integrants 1 Transfect your mammalian host cell line with your pcDNA 3 1 construct using the desired protocol Remember to include a plate of untransfected cells as a negative control and the pcDNA 3 1 CAT plasmid as a positive control 24 hours after transfection wash the cells and add fresh medium to the cells 48 hours after transfection split the cells into fresh medium containing Geneticin at the pre determined concentration required for your cell line Split the cells such that they are no more than 25 confluent 4 Feed the cells with selective medium every 3 4 days until Geneticin resistant foci can be identified 5 Pick and expand colonies in 96 or 48 well plates Appendix pcDNATM3 1 Vectors Map 10 The figure below summarizes the features of the pcDNA 3 1 and pcDNA 3 1 vectors The complete sequences for pecDNA 3 1 and pcDNA 3 1 are available for down loading from our World Wide Web site www invitrogen com or from Technical Support see page 13 Details of the
12. cells must be clean and free contamination with 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 HQ Mini Plasmid Purification Kit Catalog no K2100 01 the PureLink HiPure Plasmid Midiprep Kit Catalog no K2100 04 or CsCl gradient centrifugation 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 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 For high efficiency transfection in a broad range of mammalian cell lines we recommend using Lipofectamine 2000 Reagent Catalog no 11668 027 available from Invitrogen For more information about Lipofectamine 2000 and other transfection reagents refer to our Web site www invitrogen com or contact Technical Support page 13 TM pcDNA 3 1 CAT is provided as a positive control
13. ch 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 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 Boshart M Weber F Jahn G Dorsch H sler K Fleckenstein B and Schaffner W 1985 A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus Cell 41 521 530 Chen C and Okayama H 1987 High Efficiency Transformation of Mammalian Cells by Plasmid DNA Mol Cell Biol 7 2745 2752 Chu G Hayakawa H and Berg
14. com or by contacting Technical Support see page 13 ATG CAT Comments for pcDNA3 1 CAT 6217 nucleotides CMV promoter bases 232 819 T7 promoter priming site bases 863 882 CAT ORF bases 1027 1686 pcDNA3 1 BGH reverse priming site bases 1811 1828 BGH polyadenylation sequence bases 1817 2041 f1 origin bases 2087 2515 SV40 early promoter and origin bases 2520 2863 Neomycin resistance gene ORF bases 2925 3719 SV40 early polyadenylation sequence bases 3893 4023 pUC origin bases 4406 5076 complementary strand Ampicillin resistance gene ORF bases 5221 6081 complementary strand Technical Support World Wide Web 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 Complete technical support contact information e Access to the Invitrogen Online Catalog 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 Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan K K Invitrogen Ltd 5791 Van Allen Way Nihonbashi Hama Cho Park Inchinnan Business Park Carlsbad CA 92008 USA Bldg 4F 3 Fountain Drive Tel 1 760 603 7200 2 35
15. ible Be sure that your insert does not contain the restriction enzyme site you wish to use to linearize your vector Enzyme Restriction Site Location Supplier bp Bel II 12 Upstream of CMV Invitrogen Catalog no 15213 028 promoter Mfel 161 Upstream of CMV New England Biolabs promoter Bst1107 I 3236 End of SV40 polyA AGS Fermentas Takara Roche Mol Biochemicals Eam1105 I 4505 Ampicillin gene AGS Fermentas Takara Poul 4875 Ampicillin gene Invitrogen Catalog no 25420 019 Sca I 4985 Ampicillin gene Invitrogen Catalog no 15436 017 Ssp I 5309 bla promoter Invitrogen Catalog no 15458 011 Angewandte Gentechnologie Systeme Possible Sites for Linearizatio of pcDNA 3 1 The table below lists unique restriction sites that may be used to linearize your N pcDNA 3 1 construct prior to transfection Other unique restriction sites are possible linearize your vector Be sure that your insert does not contain the restriction enzyme site you wish to use to Enzyme Restriction Site Location Supplier bp Bgl II 12 Upstream of CMV Invitrogen Catalog no 15213 028 promoter Mfe I 161 Upstream of CMV New England Biolabs promoter Bst1107 I 3235 End of SV40 polyA AGS Fermentas Takara Roche Mol Biochemicals Eam1105 I 4504 Ampicillin gene AGS Fermentas Takara Poul 4874 Ampicillin gene Invitrogen Catalog no 25420 019
16. invitrogen pcDNA 3 1 pcDNATM3 1 Catalog nos V790 20 and V795 20 Version K 10 November 2010 28 0104 11 Table of Contents Important OIM ON AA AAA AAA v ECOS ELO ES AA ea via E E A ea vi Methods cr aso 1 DO NN eG Roe 1 Cloning into PeDNA MI Eee 2 Transtech A O A es 6 CrestingstabletellLmes ae 7 Appendix aaa 10 BEDNAPS T Meco iMpenVecnienne 10 PCDI MAA ad 12 Technical Supporto cassia acute a eee a E 13 Purchaser Noticas aa i a ia Eina aiiin 13 References ke eh O 15 iii iv Important Information pcDNA Vectors Shipping and Storage Contents Product Qualification This manual is supplied with the following products Product Catalog no pcDNA 3 1 Vector V790 20 pcDNA 3 1 Vector V795 20 Vectors are shipped on wet ice Upon receipt store at 20 C TM The pcDNA 3 1 vector components peDNA 3 1 are listed below Item Concentration Volume pcDNA 3 1 Vector 20 ug at 0 5 1g pl in TE buffer pH 8 0 40 ul pcDNA 3 1 or 10 mM Tris HCI 1 mM EDTA pH 8 0 pceDNA 3 1 Control Plasmid 20 ug at 0 5 ug ul in TE buffer pH 8 0 40 ul pcDNA 3 1 CAT 10 mM Tris HCI 1 mM EDTA pH 8 0 The Certificate of Analysis provides detailed quality control 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 l
17. mmend that you linearize your pcDNA 3 1 construct before transfection While linearizing the vector may not improve the efficiency of transfection it increases the chances that the vector does not integrate in a way that disrupts elements necessary for expression in mammalian cells To linearize your construct cut at a unique site that is not located within a critical element or within your gene of interest Geneticin blocks protein synthesis in mammalian cells by interfering with ribosomal function It is an aminoglycoside similar in structure to neomycin gentamycin and kanamycin Expression in mammalian cells of the bacterial aminoglycoside phosphotransferase gene APH derived from Tn5 results in detoxification of Geneticin Southern and Berg 1982 To successfully generate a stable cell line expressing your protein of interest you need to determine the minimum concentration of Geneticin required to kill your untransfected host cell line Test a range of concentrations see protocol below to ensure that you determine the minimum concentration necessary for your cell line 1 Plate or split a confluent plate so the cells will be approximately 25 confluent Prepare a set of 6 7 plates Add the following concentrations of antibiotic to each plate e For Geneticin selection test 0 50 125 250 500 750 and 1000 ug ml Geneticin 2 Replenish the selective media every 3 4 days and observe the percentage of surviving cells
18. o 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 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 whi
19. ond Edition Plainview New York Cold Spring Harbor Laboratory Press Shigekawa K and Dower W J 1988 Electroporation of Eukaryotes and Prokaryotes A General Approach to the Introduction of Macromolecules into Cells BioTechniques 6 742 751 Southern P J and Berg P 1982 Transformation of Mammalian Cells to Antibiotic Resistance with a Bacterial Gene Under Control of the SV40 Early Region Promoter J Molec Appl Gen 1 327 339 Wigler M Silverstein S Lee L S Pellicer A Cheng Y C and Axel R 1977 Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells Cell 11 223 232 1997 2008 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal of human therapeutic or diagnostic use 15 invitrogen Corporate Headquarters Invitrogen Corporation 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
20. ot number which is printed on the box Accessory Products Additional Additional products that may be used with the pcDNA 3 1 vectors are available from Products Invitrogen Ordering information is provided below Product Amount Catalog no One Shot TOP10 Chemically 10 reactions C4040 10 Competent Cells 20 reactions C4040 03 One Shot TOP10F Chemically 20 reactions C3030 03 Competent Cols 40 reactions 3030 06 Lipofectamine 2000 1 5 ml 11668 019 0 75 ml 11668 027 Geneticin 1g 11811 023 58 11811 031 PureLink HQ Mini Plasmid 100 preps K2100 01 Purification Kit PureLink HiPure Plasmid 25 preps K2100 04 Midiprep Kit vi Methods Overview Description Experimental Outline pcDNA 3 1 and pcDNA 3 1 are 5 4 kb vectors derived from pcDNA 3 and designed for high level stable and transient expression in mammalian hosts High level stable and non replicative 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 Multiple cloning sites in the forward and reverse orientations to facilitate cloning e Neomycin resistance gene for selection of stable cell lines e Episomal replication in cells lines that are latently infected with SV40 or that express the SV40 large T antigen e g COS 1
21. se orientation Allows insertion of your gene and facilitates cloning Bovine growth hormone BGH Efficient transcription termination and polyadenylation signal polyadenylation of mRNA Goodwin and Rottman 1992 fl origin Allows rescue of single stranded DNA SV40 early promoter and origin Allows efficient high level expression of the neomycin resistance gene and episomal replication in cells expressing SV40 large T antigen Neomycin resistance gene Selection of stable transfectants in mammalian cells Southern and Berg 1982 SV40 early 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 Ampicillin bla resistance gene B lactamase Allows selection of transformants in E coli 11 pcDNA 3 1 CAT Description Map 12 pcDNA 3 1 CAT is a 6217 bp control vector containing the gene for CAT It was constructed by digesting peDNA 3 1 with Xho I and Xba I and treating with Klenow An 800 bp Hind III fragment containing the CAT gene was treated with Klenow and then ligated into pcDNA 3 1 The figure below summarizes the features of the peDNA 3 1 CAT vector The complete nucleotide sequence for peDNA 3 1 CAT is available for downloading from our World Wide Web site www invitrogen
22. structions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Support Representatives Invitrogen assumes no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose continued on next page Purchaser Notification 13 Introduction Limited Use Label License No 5 Invitrogen Technology 14 Use of pcDNA 3 1 is covered under the licenses detailed below 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 components or c materials made using this product or its components t
23. tiation sequence with an ATG initiation codon for proper initiation of translation Kozak 1987 Kozak 1991 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 AJNNATGG Your insert must also contain a stop codon for proper termination of your gene Please note that the Xba I site contains an internal stop codon TCTAGA continued on next page Cloning into pcDNATM3 1 continued Multiple Cloning Site of pcDNA 3 1 749 809 869 929 989 1049 1109 BamHI Below is the multiple cloning site for p DNA 3 1 Restriction sites are labeled to indicate the cleavage site The Xba I site contains an internal stop codon TCTAGA The multiple cloning site has been confirmed by sequencing and functional testing The complete sequence of pcDNA 3 1 is available for downloading from our web site www invitrogen com or from Technical Support see page 13 For a map and a description of the features of pcDNA 3 1 please refer to the Appendix pages 10 11 enhancer region 3 end CATTGACGTC AATGGGAGTT TGTTTTGGCA CCAAAATCAA CGGGACTTTC CAAA

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