pcDNA4/TO-E - Thermo Fisher Scientific
Contents
1. Cell Line Source Catalog no T REx 293 Human embryonic kidney R710 07 T REx HeLa Human cervical adenocarcinoma R714 07 T REx CHO Chinese hamster ovary cells R718 07 T REx Jurkat Human lymphocyte R722 07 TM Many of the reagents used in the T REx System are available separately from Invitrogen See the table below for ordering information Item Amount Catalog no pcDNA6 TR 20 ug lyophilized V1025 20 Tetracycline 5 g powder Q100 19 Zeocin lg R250 01 5g R250 05 Blasticidin 50 mg powder R210 01 Continued on next page Related Products Continued Additional Products Donor Vectors Many of the reagents supplied with the pe DNA4 TO E Echo Adapted Expression Vector as well as additional reagents that may be used with the pcDNA4 TO E are available separately from Invitrogen Ordering information is provided below Product Amount Catalog no PIRI One Shot E coli chemically competent 11x50 ul C1010 10 Cre Recombinase 10 reactions R100 10 Anti His C term Antibody 50 ul R930 25 Anti His C term HRP Antibody 50 pl R931 25 Anti V5 Antibody 50 ul R960 25 Anti V5 HRP Antibody 50 ul R961 25 ProBond Purification System 6 purifications K850 01 ProBond Purification System with Anti 1 kit K853 01 His C term HRP Antibody ProBond Purification System with Anti V5 HRP 1 kit K854 01 Antibo2. Adapted Expression Vector Kit Reagents Supplied Catalog nos pcDNA4 TO E Echo Adapted pcDNA4 TO E vector ET460 01 Expression Vector Kit Expression control vector Cre Recombinase and 10X buffer CMV Forward Primer pcDNA4 TO E Echo Adapted pUni V5 His TOPO TA Cloning Kit ET460 10C Expression Vector Kit with a choice pUpiBlunt V5 His TOPO Cloning Kit ET460 20C of Donor Vector Kit and One Shot TOP10 Chemically Competent pUni V5 His A B and C ET460 30C E coli see page 25 for more pUniD V5 His TOPO Cloning Kit ET460 40C information on donor vectors Shipping Storage The pcDNA4 TO E Echo Adapted Vector Kit is shipped on dry ice Upon receipt store the p DNA4 TO E reagents at 20 C Store the One Shot Competent E coli at 80 C pcDNA4 TO E The peDNA4 TO ETeagents are listed below Store at 20 C Reagents Item Concentration Amount pcDNA4 TO E Supercoiled lyophilized in TE pH 8 0 20 ug Cre Recombinase Please check the label on the tube for exact 12 ul concentration of the enzyme Enzyme supplied in 50 mM Tris HCl pH 8 0 5 mM EDTA 1 mM EGTA 10 mM mercaptoethanol 20 Glycerol 10X Recombinase Buffer 500 mM Tris HCl pH 7 5 25 ul 100 mM MgCl 300 mM NaCl 1 0 mg ml BSA CMV Forward Primer Lyophilized in TE Buffer pH 8 0 2 ug 21 mer 5 CGCAAATGGGCEGGTAGGCGTG 3 306 pmoles Expression Control Supercoiled lyophil
3. Bacteriophage P1 Site Specific Recombination Purification and Properties of the Cre Recombinase Protein J Biol Chem 259 1509 1514 Abremski K Hoess R and Sternberg N 1983 Studies on the Properties of P1 Site Specific Recombination Evidence for Topologically Unlinked Products Following Recombination Cell 32 1301 1311 Abremski K E and Hoess R H 1992 Evidence for a Second Conserved Arginine Residue in the Integrase Family of Recombination Proteins Protein Eng 5 87 91 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 Argos P Landy A Abremski K Egan J B Haggard Ljungquist E Hoess R H Kahn M L Kalionis B Narayana S V L Pierson III L S Sternberg N and Leong J M 1986 The Integrase Family of Site Specific Recombinases Regional Similarities and Global Diversity EMBO J 5 433 440 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
4. Pl Site Specific Recombination Nucleotide Sequence of the Recombining Sites Proc Natl Acad Sci USA 79 3398 3402 Liu Q Li M Z Leibham D Cortez D and Elledge S 1998 The Univector Plasmid Fusion System a Method for Rapid construction of Recombinant DNA Without Restriction Enzymes Current Biology 8 1300 1309 Continued on next page 29 References Continued Liu Q Li M Z Liu D and Elledge S J 1999 Rapid Construction of Recombinant DNA by the Univector Plasmid Fusion System Methods in Enzymology in press 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 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 Harbor Laboratory Press Sternberg N Hamilton D Aust
5. Antibiotic Sensitivity Once you have established that your construct can be inducibly expressed you may create a stable cell line that carries the tet repressor and inducibly expresses your gene of interest p DNA4 TO E contains the Zeocin resistance gene to allow selection of stable lines using Zeocin pcDNA6 TO carries the blasticidin resistance gene Before establishing a double stable cell line it is important to determine the minimum concentration of selection agents required to kill untransfected cells The protocol below provides information for determining the appropriate concentration of Zeocin A similar TM protocol for blasticidin can be found in the T REx System manual Please note that your gene of interest will be constitutively expressed if you transfect your pcDNA4 TO E fusion vector into mammalian host cells prior to transfecting the pcDNA6 TR plasmid For more information on selection of stable cell lines using pcDNAG TR and blasticidin please refer to the T REx System manual Reminder When generating a stable cell line expressing the Tet repressor from pcDNAG6 TR you will want to select for clones that express the highest levels of Tet repressor to use as hosts for your peDNA4 TO E fusion vector Those clones that express the highest levels of Tet repressor should exhibit the most complete repression of basal transcription of your gene of interest To generate a stable cell line expressing
6. 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 Continued on next page Purchaser Notification Continued Limited Use Label No license is conveyed to use this product with any recombination sites other License than those purchased from Life Technologies Corporation or its authorized No 119 Echo distributor The buyer cannot modify the recombination sequence s contained Cloning Products in this product for any purpose 27 Technical Service Web Resources Visit the Invitrogen Web site at www invitrogen com for e Technical resources including manuals vector maps and
7. 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 47 521 530 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 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 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 Gossen M and Bujard H 1992 Tight Control of Gene Expression in Mammalian Cells by Tetracycline Responsive Promoters Proc Natl Acad Sci USA 89 5547 5551 Hillen W and Berens C 1994 Mechanisms Underlying Expression of Tn 0 Encoded Tetracycline Resistance Annu Rev Microbiol 48 345 369 Hillen W C G Altschmied L Schollmeier K and Meier I 1983 Control of Expression of the Tn 0 Encoded Tetracycline Resistance Genes Equilibrium and Kinetic Investigations of the Regulatory Reactions J Mol Biol 169 707 721 Hoess R H Ziese M and Sternberg N 1982
8. TetO operator sequence in pcDNA4 TO E to maximize repression of basal gene expression For more detailed information about tet operators please refer to Hillen and Berens 1994 Yao et al 1998 have recently demonstrated that the location of tet operator sequences in relation to the TATA box of a heterologous promoter is critical to the function of the tet operator Regulation by tetracycline is only conferred upon a heterologous promoter by proper spacing of the TetO sequences from the TATA box Yao et al 1998 For this reason the first nucleotide of the TetO operator sequence has been placed 10 nucleotides after the last nucleotide of the TATA element in the CMV promoter in pcDNA4 TO E Please refer to the diagram on page 8 for the sequence and placement of the TetO sequences in relation to the TATA box In other tetracycline regulated systems the TetO sequences are located upstream of the TATA element in the promoter of the inducible expression vector Gossen and Bujard 1992 These systems differ substantially from the T REx System in that they use regulatory molecules composed of the Tet repressor fused to a viral transactivation domain The presence of viral transactivation domains appears to overcome the requirement for specific positioning of the TetO sequences in relation to the TATA box of the heterologous promoter However the presence of viral transactivation domains has been found to have deleterious effects in some
9. V1025 20 For more information about pcDNA6 TR and the T REx System please refer to the T REx System manual our World Wide Web site www invitrogen com or call Technical Service see page 29 The Echo Cloning System is based on the univector plasmid fusion system UPS des cribed by Elledge and coworkers to quickly and easily recombine a gene of interest into a series of recipient acceptor vectors Liu et al 1998 Liu et al 1999 The system con sists of the univector donor vector containing the gene of interest and recipient accep tor vectors containing various regulatory sequences for expression in the host of choice The Echo System utilizes the cre lox site specific recombination system of bacterio phage P1 Abremski et al 1983 Sternberg et al 1981a The product of the cre gene is a site specific recombinase that catalyzes conservative recombination between two 34 bp loxP or loxH sequences to resolve P1 dimers generated by replication of circular lysogens The donor vector and the acceptor vector i e p DNA4 TO E each contains a lox site The donor vector pUni contains a oxP site while the acceptor vector contains either a loxP or a loxH site see the next page for more information about loxH You have already constructed the donor vector containing the PCR product of interest via the TOPO Cloning method peDNA4 TO E allows you to regulate expression of your PCR product using the T REx
10. bases 820 859 loxH site bases 984 1017 BGH polyadenylation sequence bases 1049 1273 f1 origin bases 1319 1747 SV40 early promoter and origin bases 1752 2095 EM7 promoter bases 2137 2203 Zeocin resistance gene bases 2204 2578 SV40 early polyadenylation sequence bases 2708 2838 pUC origin bases 3221 3891 complementary strand bla promoter bases 4897 4995 complementary strand Ampicillin bla resistance gene bases 4036 4896 complementary strand Continued on next page pcDNA4 TO E Vector Continued Features of peDNAMT O E 5032 bp contains the following elements All features have been pcDN A4 TO E functionally tested Feature Benefit Human cytomegalovirus CMV immediate early promoter Permits high level expression of your gene of interest Andersson et al 1989 Boshart et al 1985 Nelson et al 1987 CMV Forward priming site Allows sequencing in the sense orientation Tetracycline operator O2 sequences Two tandem 19 nucleotide repeats which serve as binding sites for Tet repressor homodimers Hillen and Berens 1994 loxH site Allows recombination between the donor vector and pe DNA4 TO E Hoess et al 1982 BGH reverse priming site Permits sequencing through the insert Bovine growth hormone BGH polyadenylation sequence Permits efficient transcription termination and polyadenylation of mRNA Goodwin and Rottman 1992 fl
11. sequences application notes MSDSs FAQs formulations citations handbooks etc e Complete technical service 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 Web page www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters Invitrogen Corporation Invitrogen Japan Invitrogen Ltd 1600 Faraday Avenue 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 E mail E mail tech_service invitrogen com jpinfo invitrogen com eurotech invitrogen com Material Data Safety Sheets MSDSs Limited Warranty 28 MSDSs are available on our Web site at www invitrogen com On the home page click on Technical Resources and follow instructions on the page to download the MSDS for your product 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 Invitr
12. your protein of interest you need to determine the minimum concentration of Zeocin required to kill your untransfected host cell line Typically concentrations between 50 and 1000 ug ml Zeocin are sufficient to kill the untransfected host cell line Test a range of concentrations see below to ensure that you determine the minimum concentration necessary for your cell line For more information on Zeocin including instructions on preparation and storage please refer to page 21 Note Before transfecting your host cell line with pcDNA6 TR you will need to perform a similar experiment to determine the minimum concentration of blasticidin required to kill the untransfected cell line Please refer to the T REx System manual for information about blasticidin e Plate or split a confluent plate so the cells will be approximately 25 confluent Prepare a set of 7 plates e The next day substitute culture medium with medium containing varying concentrations of Zeocin e g 0 50 125 250 500 750 and 1000 ug ml e Replenish the selective medium every 3 4 days and observe the percentage of surviving cells e Count the number of viable cells at regular intervals to determine the appropriate concentration of Zeocin that prevents growth within 1 2 weeks after addition of TM Zeocin 13 Creation of Stable Cell Lines Continued Effect of Zeocin on Sensitive and Resistant Cells Plasmid Linearization Sele
13. LB and SOB medium 2 Isolate plasmid DNA using your method of choice If you need ultra pure plasmid DNA for automated or manual sequencing we recommend the S N A P MiniPrep Kit 10 15 ug DNA Catalog no K1900 01 or the S N A P MidiPrep Kit 10 200 ug DNA Catalog no K1910 01 3 Analyze the plasmids by restriction analysis Use an enzyme or enzymes that cut once in the donor vector and once in the acceptor vector to yield two fragments that are distinguishable from one another Please note that other strategies are possible 4 Optional To sequence the fusion plasmid to confirm the fusion junctions use the CMV Forward and Unil Forward sequencing primers Please refer to the diagram on the following page for the sequence around the p DNA4 TO E loxH site Refer to the donor vector manual for the sequence around the donor vector loxP site If you need help with setting up restriction enzyme digests or DNA sequencing please refer to general molecular biology texts Ausubel et al 1994 Sambrook et al 1989 Transforming the Recombination Reaction Continued Sequencing Your The sequence surrounding your insert is shown below Unique restriction sites are labeled Construct to indicate the cleavage site Please note that the complete sequence of pcDNA4 TO E is 721 791 861 931 1001 available for downloading from our Web site www invitrogen com or from Technical Service CMV Forward priming sit
14. Map of Expression The figure below summarizes the features of the pcDNA4 T O E Uni lacZ vector The Control Vector 20 complete nucleotide sequence for peDNA4 TO E Uni lacZ is available for downloading from our World Wide Web site www invitrogen com or by contacting Technical Service see page 29 pcDNA4 TO E Uni lacZ 10405 bp Comments for pcDNA4 TO E Uni acZ 10405 nucleotides CMV promoter bases 232 958 TATA box bases 804 810 Tetracycline operator 2 2X TetO sequences bases 820 859 loxH site bases 984 1017 LacZ ORF bases 1041 4097 BGH polyadenylation sequence bases 4260 4468 Kanamycin promoter bases 5585 5722 complementary strand Kanamycin resistance gene bases 4790 5584 complementary strand R6Ky origin bases 5940 6342 loxP site bases 6357 6390 BGH polyadenylation sequence bases 6422 6646 f1 origin bases 6692 7120 SV40 early promoter and origin bases 7125 7468 EM7 promoter bases 7510 7576 Zeocin resistance gene bases 7577 7951 SV40 early polyadenylation sequence bases 8081 8211 pUC origin bases 8594 9264 complementary strand bla promoter bases 10270 10368 complementary strand Ampicillin b a resistance gene bases 9409 10269 complementary strand Zeocin Zeocin Molecular Weight Formula and Structure Applications of Zeocin TM Zeocin belongs to a family of structurally related bleomycin phleomycin type antibiotics isolated from Streptomyce
15. System The unique loxH site is located downstream of the regulatory sequences By mixing the donor vector containing the PCR product of interest with pcDNA4 TO E in the presence of Cre recombinase a plasmid fusion is created that expresses the PCR product in mammalian cells A generic diagram is shown below pUni 2 3 kb gene Po O Recombinant Plasmid 4 8 kb gene to 8 1 kb gene pAcceptor 2 5 to 5 8 kb lox loxP or loxH depending on acceptor vector Continued on next page Overview Continued loxP or loxH Sites Cre Recombinase Selection of Recombinants pcDNA4 TO E The sequence of the oxP site is shown below The loxP site consists of a 34 bp sequence containing two 13 bp inverted repeats see underlined bases separated by an 8 bp spacer Hoess et al 1982 The inverted repeats may form a stem and loop structure that may reduce expression of the gene of interest in some cases A variation of the loxP site loxH see below was created to eliminate the formation of a stem and loop structure and improve expression Mutated bases are shown in boldface Please note that some acceptor vectors including peDNA4 TO E contain a oxH site Cre mediated recombination can still occur between a loxP and a loxH site although the efficiency may be slightly reduced e loxP ATA ACT TCG TAT AGC ATA CAT TAT ACG AAG TTA T e loxH ATT ACC TCA TAT AGC ATA CAT TAT ACG AAG TTA T Cre rec
16. age 1 2 3 4 5 Streak out the original colony on LB plates containing 50 ug ml kanamycin to isolate single colonies Select a single colony and inoculate into 1 2 ml of LB containing 50 ug ml kanamycin Grow overnight until culture is saturated Mix 0 85 ml of culture with 0 15 ml of sterile glycerol and transfer to a cryovial Store at 80 C You may also want to store a stock of plasmid DNA at 20 C Transfection and Analysis Introduction Plasmid Preparation Important Cotransfection and Induction with Tetracycline 10 Once you have created the pcDNA4 TO E fusion plasmid have verified its integrity and have prepared clean plasmid preparations of both the fusion plasmid and pceDNA6 TR you are ready to cotransfect the plasmids into the mammalian cell line of choice Please refer to the T REx System manual for complete information on pcDNA6 TR transfection and induction of expression We recommend that you include the positive expression control vector and a mock transfection negative control 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 the transfection efficiency We recommend isolating plasmid DNA using the S N A P MidiPrep Kit Catalog no K1910 01 or other resin based method Because tetracycline regulated ex
17. at is selected on kanamycin will represent a recombined fusion plasmid pcDNA4 TO E is a 5 0 kb vector derived from pcDNA4 TO and designed for high level inducible expression in most mammalian hosts The vector contains the following elements e Cytomegalovirus CMV immediate early promoter containing two tetracycline operator 2 TetO 2 sites for tetracycline regulated expression of your gene of interest in mammalian cells Yao et al 1998 e A loxH site for univector plasmid fusion e Zeocin resistance gene for selection of stable cell lines Mulsant er al 1988 e The pUC origin for high copy replication and maintenance in most E coli strains e The ampicillin bla resistance gene for selection in E coli For a map and a description of peDNA4 TO E please refer to page 18 Other Echo adapted acceptor vectors are available separately and are provided with their own manuals For more information on other available acceptor vectors please visit our Web site www invitrogen com or call Technical Service see page 29 Continued on next page Overview Continued Repression and Derepression Tet Operator Sequences The TetO sequences in the peDNA4 TO E vector serve as binding sites for four Tet repressor molecules comprising two Tet repressor homodimers and confer tetracycline responsiveness to your gene of interest The Tet repressor is expressed from the pceDNA6 TR plasmid For more information abou
18. bsence of phage contamination 0 5 1 ml of competent cells are added to LB top agar and poured onto LB plates After overnight incubation no plaques should be detected e Untransformed cells are plated on LB plates 100 ug ml ampicillin 25 ug ml streptomycin 50 ug ml kanamycin or 15 ug ml chloramphenicol to verify the absence of antibiotic resistant contamination 23 Related Products Additional Products T REx Echo Expression System Core Kit T REx Cell Lines T REx System Components 24 The T REx Echo Expression Support Kit and individual reagents for inducible expression of your gene of interest from the peDNA4 TO E fusion vector are available from Invitrogen Ordering information is provided below The T REx Echo Expression System Support Kit contains the pcDNA6 TR vector which expresses the Tet repressor Zeocin blasticidin tetracycline and a comprehensive instruction manual detailing the procedure for inducible expression in mammalian cell lines Item Catalog no TM TM T REx Echo Expression System Support Kit K1020 03 For your convenience Invitrogen offers four mammalian cell lines that stably express the Tet repressor Expression of your gene of interest from pcDNA4 TO may be assayed by transfection of your pcDNA4 TO construct into any of the T REx cell lines and induction with tetracycline Ordering information is provided below
19. ction of Stable Integrants 14 The method of killing of Zeocin is quite different from blasticidin neomycin and hygromycin Cells do not round up and detach from the plate Sensitive cells 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 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 For more information about Zeocin please see page 21 We have found that it is not necessary to linearize the peDNA4 TO E fusion vector prior to transfection Once you have determined the appropriate Zeocin concentration to use for selection you can generate a stable cell line expressing pcDNA6 TR and your pcDNA4 TO E fusion vector We recommend that you first generate a stable cell line expressing pcDNA6 TR and then use this cell line as the host for your ppDNA4 TO E fusion vect
20. dy ProBond Metal Binding Resin 50 ml R801 01 150 ml R801 15 Purification Columns 50 R640 50 One Shot TOP10 chemically competent E coli 21x50 ul C4040 03 Quantity supplied is sufficient for 25 western blots The table below lists a variety of donor vectors currently available from Invitrogen to facilitate cloning of your gene of interest for use with Echo Cloning System Product Application Quantity Catalog no pUni V5 His TOPO TA Cloning A tailed PCR products 10 reactions ETOO1 10 Cloning Kit pUniBlunt V5 His TOPO Cloning blunt PCR products 10 reactions ET002 10 Cloning Kit pUniD V5 His TOPO Directional cloning of blunt PCR 10 reactions ET004 10 Cloning Kit products pUni V5 His A B and C Cloning DNA fragments using 10 reactions ET003 10 restriction enzymes 25 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology Limited Use Label License No 22 Vectors and Clones Encoding Histidine Hexamer 26 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
21. e I l AAAATCAACG GGACTTTCCA AAATGTCGTA ACAACTCCGC CCCATTGACG CAAATGGGCG GTAGGCGTGT TATA box Tetracycline operator TetO Tetracycline operator TetO2 El Co ee ACGGTGGGAG GTCTATATAA GCAGAGCTCT CCCTATCAGT GATAGAGATC TCCCTATCAG TGATAGAGAT CGTCGACGAG CTCGTTTAGT GAACCGTCAG ATCGCCTGGA GACGCCATCC ACGCTGTTTT GACCTCCATA mm GAAGACACCG GGACCGATCC AGCCTCCGGA CTCTAGCGTT TAAACTTAAG CTT ATT ACC TCA loxH site ae Gene of C terminal ta TAT AGC ATA CAT TAT ACG AAG TTA T GA g donor vector Uni1 Forward priming site gt Apal Pmel l I AGCCTCGACT GTGCCTTCTA GTTGCCAGCC ATCTGTTGTT TGCCCCTCCC CCGTGCCTTC Continued on next page Transforming the Recombination Reaction Continued Fusion Vector Analysis Preparing a Glycerol Stock for Long Term Storage It should be clear from restriction analysis that you have a dimer plasmid consisting of the donor vector and p DNA4 TO E Occasionally trimers will result Trimers usually consist of two donor vector molecules and one acceptor molecule In theory trimers may result from two sequential fusion events or a single fusion event between a pre existing monomeric substrate and a dimeric substrate The production of trimers can be eliminated if gel purified monomeric supercoiled DNA is used in the recombination reaction Please note that trimers usually express as well as the dimer product Once you have identified the correct clone prepare a glycerol stock for long term stor
22. e regulated expression of your gene of interest If you have expressed your protein as a fusion to the C terminal polyhistidine 6xHis tag you can purify it using ProBond Resin or other metal chelating resin Please refer to the manufacturer s instructions before attempting to purify your fusion protein Use the procedure below to prepare cells for lysis if you will be purifying your protein on ProBond Resin You will need 5 x 10 to 1 x 10 stably transfected cells for purification of your protein on a 2 ml ProBond column see ProBond Protein Purification manual Seed cells in either five T 75 flasks or 2 to 3 T 175 flasks 2 Grow the cells in selective medium until they are approximately 80 confluent 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 microcentrifuge tube Centrifuge the cells at 1500 rpm for 5 minutes Resuspend the cell pellet in PBS Centrifuge the cells at 1500 rpm for 5 minutes You may lyse the cells immediately or freeze in liquid nitrogen and store at 70 C until needed If you are using ProBond resin refer to the ProBond Protein Purification manual for details about sample preparation for chromatography If you are using other metal chelating resin please refer to the manufacturer s instructions for recommendations on sa
23. ern blot you may use an antibody to B galactosidase If you have expressed your protein as a fusion to the C terminal V5 epitope and polyhistidine 6xHis tag you can detect expression using the Anti V5 or Anti His C term antibodies see page 25 for ordering information You may also use an antibody to your protein of interest To detect your 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 your fusion protein e g 12 24 48 72 hours etc after transfection Use the protocol below to lyse cells Other protocols and lysis buffers are also suitable 1 Wash cell monolayers 5 x 10 to 1 x 10 cells once with phosphate buffered saline PBS see recipe on the next page 2 Scrape cells into 1 ml PBS and pellet the cells at 1500 x g for 5 minutes 3 Resuspend pellet in 50 ul Cell Lysis Buffer see recipe on the next page and vortex 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 protei
24. hours before induction e Remove medium and add fresh medium containing the appropriate concentration of tetracycline to the cells In general we recommend that you add tetracycline to a final concentration of 1 ug ml 5 ul of a 1 mg ml stock solution per 5 ml of medium to the cells and incubate the cells for 24 hours at 37 C e Harvest the cells and assay for expression of your gene of interest Continued on next page Transfection and Analysis Continued Positive Expression Control Assay for B galactosidase Activity Detection of B Galactosidase Detection of Recombinant Fusion Proteins pcDNA4 TO E Uni lacZ is provided as a positive control vector for mammalian cell transfection and expression and may be used to optimize transfection conditions for your cell line Cotransfection of the positive control vector and peDNA6 TR results in the expression of B galactosidase following the addition of tetracycline A successful cotransfection will result in positive B galactosidase expression and can be easily assayed by staining with X gal 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 Catalog no K1455 01 and the B Gal Staining Kit Catalog no K1465 01 for fast and easy detection of B galactosidase expression If you wish to detect expression of B galactosidase by west
25. in S Yarmolinsky M and Hoess R 1981a Site Specific Recombination and its Role in the Life Cycle of P1 CSH Symp Quant Biol 45 297 309 Yao F Svensjo T Winkler T Lu M Eriksson C and Eriksson E 1998 Tetracycline Repressor tetR Rather than the tetR Mammalian Cell Transcription Factor Fusion Derivatives Regulates Inducible Gene Expression in Mammalian Cells Hum Gene Ther 9 1939 1950 1999 2006 2010 Invitrogen Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use 30 Notes 31 32 8 invitrogen Corporate Headquarters Invitrogen Corporation 1600 Faraday Avenue Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500 E tech service invitrogen com For country specific contact information visit our web site at www invitrogen com
26. invitrogen pcDNA4 TO E Echo Adapted Expression Vector For cloning a gene of interest using the Echo Cloning System and inducible expression in mammalian cells using the T REx System Catalog nos ET460 XX Version F 28 October 2010 25 0327 11 Table of Contents Table of Contents daa eames iii Kit Contents and Storage iiem ea e anaE E his EEE EEEE A EEEE Eaa E EEEE v INtrOdUCHON ii 1 OVENI Wai id AA A A a a A A AAA EA di 1 MeIh ds n usnuisunsnea innen in 5 Recombining Your Gene into peDNAATO E aneinander 5 Transforming the Recombination Reaction uunsessersessersessesnnennennsnensennnnnonsennnesonnnenenenonsennonsonsnenonsonsnensenonon 6 Transfection and Analysis 4er le age ae sea di 10 Creation of Stable Cell Eines u 2 00 08 dovadsshbevicernccsteess sontetenus cuactitedscedtiodsecbuders cevehe huewieon ended varesinn es 13 PA PSII anne nissen 16 RECIPES a 16 PENA Vene Rn ne 18 I DNAFTO BilnElaez een ee Ge 20 e Be 21 Product Qualifications cots tease eA EE 23 Related Products 2 22 52 2 Ran RRR A aaa 24 Purchaser Notification c ccccceeccessceseceseeesecsnecacecseeesecceeseeesecesecseeeeeeseseseesecsaeceaeceaecaaecaeeaeeeaeeeseeeeeeeeeeereeereeaees 26 Technical AV das 29 References NRO 30 111 1v Kit Contents and Storage Types of Kits Several p DNA4 TO E Echo Cloning System Kits are available The table below lists the kits that include the p DNA4 TO E Echo
27. ion Enzyme pcDNA4 TO E pcDNA4 TO E Uni lacZ Avr II linearizes 5032 bp Not tested Bgl Il 4208 bp 824 bp Not tested Hind Il 5032 bp 5327 bp 5078 bp BamH I linearizes Not tested 10405 bp Pme 1 Not tested 5429 bp 4976 bp The CMV Forward Sequencing Primer has been lot qualified by DNA sequencing experiments using the dideoxy chain termination technique Purity gt 95 homogeneity Endonuclease activity Negative Exonuclease activity Negative Functional Assay Cre recombinase is qualified using the assay on page 23 of this manual The donor vector is pUni lacZ and the acceptor vector is peDNA3 1 E Five microliters of the recombination reaction is transformed into 50 ul TOP10 One Shot competent E coli using the protocol on page 7 Twenty five ul of the transformation reaction is plated on LB plates containing 50 ug ml kanamycin performed in duplicate One microliter of Cre recombinase should yield gt 500 blue kanamycin resistant transformants All competent cells are qualified as follows e Cells are tested for transformation efficiency using the control plasmid included in the kit Transformed cultures are plated on LB plates containing 100 ug ml ampicillin and the transformation efficiency is calculated Test transformations are performed in duplicate Transformation efficiency should be 1 x 10 cfu ug DNA for chemically competent cells and gt 1 x 10 for electrocompetent cells e To verify the a
28. 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 which this product or its components was employed
29. ized in TE pH 8 0 20 ug pcDNA4 TO E Uni lacZ Continued on next page Kit Contents and Storage Continued One Shot Reagents Optional Genotype of TOP10 vi The table below describes the items included in the One Shot Competent E coli kit Store at 80 C EDTA pH 8 Item Concentration Amount SOC Medium 2 Tryptone 6 ml may be stored at room 0 5 Yeast Extract temperature or at 10 mM NaCl 4 C 2 5 mM KCl 10 mM MgCl 10 mM MgSO 20 mM glucose TOP10 E coli 11x 50 ul pUC19 Control DNA 10 pg ul in 5 mM Tris HCl 0 5 mM 50 ul TOP10 Use this strain for general cloning of your gene of interest Note This strain cannot be used for growth and transformation of donor vectors F mcrA A mrr hsdRMS mcrBC p80lacZAM15 AlacX74 recAl araD139 A ara leu 7697 galU galK rpsL StrR endAl nupG Overview Introduction Echo Cloning System Plasmid Fusion Introduction TM The Echo Cloning System allows direct recombination of your gene of interest downstream of an appropriate promoter for expression in the host system of choice pcDNA4 TO E is a member of the Echo Cloning System family of expression vectors and is specifically designed for inducible expression in mammalian cells The vector allows tetracycline regulated expression of the gene of interest in mammalian host cells cotransfected with the peDNA6 TR vector Catalog no
30. mammalian cell lines Continued on next page Overview Continued Experimental The table below describes the general steps needed to recombine transform and express Outline your protein of interest Step Action Page 1 Perform the recombination reaction using your donor vector and 5 pcDNA4 TO E 2 Transform the recombination reaction into competent TOP10 E coli 6 cells Select transformants on LB plates containing 50 ug ml kanamycin 7 Analyze transformants by restriction digestion 7 5 Select the correct clone and cotransfect your construct and peDNA6 TR 10 into the mammalian cell line of interest using your method of choice 6 Induce expression of your recombinant protein with tetracycline and 10 analyze by western blot or functional assay 7 Generate a double stable cell line if desired 13 Purify your protein if desired 15 Methods Recombining Your Gene into pcDNA4 TO E Introduction Preparation and Maintenance of pcDNA4 TO E Before Starting Recombination Reaction At this point you should have a plasmid preparation of your donor vector construct in ia TM addition to p DNA4 TO E Please review the information below and on the next page before performing the recombination reaction To prepare pcDNA4 TO E for use add 20 ul sterile water to create a 1 ug ul stock solution You can further dilute a small aliquot of plasmid or use the stock solu
31. mple preparation 15 Recipes LB Luria Bertani Medium and Plates Low Salt LB Medium with Zeocin 16 Appendix Composition 1 0 Tryptone 0 5 Yeast Extract 1 0 NaCl pH 7 0 1 For 1 liter dissolve 10 g tryptone 5 g yeast extract and 10 g NaCl in 950 ml deionized water 2 Adjust the pH of the solution to 7 0 with NaOH and bring the volume up to 1 liter 3 Autoclave on liquid cycle for 20 minutes at 15 psi Allow solution to cool to 55 C and add antibiotic if needed 4 Store at room temperature or at 4 C LB agar plates 1 Prepare LB medium as above but add 15 g L agar before autoclaving 2 Autoclave on liquid cycle for 20 minutes at 15 psi 3 After autoclaving cool to 55 C add antibiotic 50 ug ml of kanamycin and pour into 10 cm plates 4 Let harden then invert and store at 4 C in the dark For Zeocin to be active the salt concentration of the medium must be low lt 90 mM and the pH must be 7 5 Use the medium below to prepare plates and liquid medium for selection in E coli Failure to use low salt LB medium will result in non selection due to inactivation of the drug 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 for 20 minutes 3 Thaw Zeocin on ice a
32. n 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 Continued on next page 11 Transfection and Analysis Continued Note Cell Lysis Buffer Phosphate Buffered Saline PBS 12 The C terminal peptide containing the V5 epitope and the polyhistidine 6xHis tag will add approximately 5 kDa to the size of your protein 50 mM Tris pH 7 8 150 mM NaCl 1 Nonidet P 40 1 2 3 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 Bring the volume up to 90 ml with deionized water and adjust the pH to 7 8 with HCl Bring the volume up to 100 ml Store at room temperature Note Just before use add protease inhibitors to a small amount of buffer at the following final concentrations 1 mM PMSF 1 ug ml pepstatin 1 ug ml leupeptin 137 mM NaCl 2 7 mM KCl 10 mM Na HPO 1 8 mM KH PO 1 Dissolve the following in 800 ml of deionized water 8 g NaCl 0 2 g KCl 1 44 g Na HPO 0 24 g KH PO Adjust pH to 7 4 with concentrated HCl Bring the volume up to 1 liter and autoclave for 20 minutes on liquid cycle Store at 4 C or room temperature Creation of Stable Cell Lines Introduction Note Determination of
33. nd vortex before removing an aliquot Allow the medium to cool to at least 55 C before adding the Zeocin to 25 50 ug ml final concentration 5 Store plates at 4 C in the dark Plates containing Zeocin are stable for 1 2 weeks Continued on next page Recipes Continued SOB Medium with SOB per liter Kanamycin 2 Tryptone 0 5 Yeast Extract 0 05 NaCl 2 5 mM KCl 10 mM MgCl 1 Dissolve 20 g tryptone 5 g yeast extract and 0 5 g NaCl in 950 ml deionized water 2 Make a 250 mM KCI solution by dissolving 1 86 g of KCl in 100 ml of deionized water Add 10 ml of this stock KCI solution to the solution in Step 1 3 Adjust pH to 7 5 with 5 M NaOH and add deionized water to liter 4 Autoclave this solution cool to 55 C and add 10 ml of sterile 1 M MgCl You may also add kanamycin to 50 ug ml 5 Store at 4 C Medium is stable for only 1 month 17 pcDNA4 TO E Vector Map of pcDNA4 TO E 18 The figure below summarizes the features of the pe DNA4 TO E vector The complete sequence for peDNA4 TO E is available for downloading from our World Wide Web site www invitrogen com or from Technical Service see page 29 Details of the sequences surrounding the loxH site in p DNA4 TO E may be found on page 8 pcDNA4 TO E 5032 bp Comments for pcDNA4 TO E 5032 nucleotides CMV promoter bases 232 958 TATA box bases 804 810 Tetracycline operator 2 2X TetO sequences
34. ng Zeocin High salt and acidity or basicity inactivate 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 Low Salt LB Medium page 16 Please note that the pH and salt concentration do not need to be adjusted when preparing tissue culture medium containing Zeocin Store Zeocin at 20 C and thaw on ice before use Zeocin is light sensitive Store the drug and plates or medium containing drug in the dark at 4 C Culture medium containing Zeocin may be stored at 4 C protected from exposure to light for up to 1 month Wear gloves a laboratory coat and safety glasses or goggles when handling Zeocin containing solutions Zeocin is toxic Do not ingest or inhale solutions containing the drug Preparing and Zeocin is available from Invitrogen see page 24 for ordering information For your Storing Zeocin convenience Zeocin is prepared in autoclaved deionized water in 1 25 ml aliquots at aco ncentration of 100 mg ml The stability of Zeocin is guaranteed for six months if stored at 20 C protected from exposure to light 22 Product Qualification Vectors Primers Cre Recombinase One Shot Competent E coli pcDNA4 TO E and pcDNA4 TO E Uni lacZ are qualified by restriction digest The table below lists the restriction enzymes and the expected fragments Restrict
35. ogen product or service contact our Technical Service 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 instructions Invitrogen reserves the right to select the method s used to analyze a product unless Invitrogen agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore Invitrogen makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications please report it to our Technical Service 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 References Abremski K and Hoess R 1984
36. ombinase MW 35 kDa is a site specific recombinase that binds to specific sequences JoxP and loxH sites brings together the target sites cleaves and covalently attaches to the DNA Recombination occurs following two pairs of strand exchanges and ligation of the DNAs in a novel recombinant form A nucleophilic hydroxylated tyrosine initiates the DNA cleavage event by attack on a specific phosphodiester bond followed by the covalent attachment of the recombinase to the target sequence through a phosphoamino acid bond Abremski and Hoess 1992 Argos ef al 1986 The reaction does not require any host factors or ATP but does require Mg or spermidine for activity Abremski et al 1983 Recombination between two supercoiled substrates each containing a loxP or loxH site results in a supercoiled dimer The extent of the reaction is 10 20 and appears to be stoichiometric Abremski and Hoess 1984 Abremski et al 1983 By fusing the two plasmids kanamycin resistance is now linked to the pUC origin of replication The recombination reaction is transformed into TOP10 E coli and recombinants selected by plating the transformation reaction onto plates containing kanamycin Because the donor plasmid carries the R6Ky origin of replication it will not propagate in E coli such as TOP10 which do not carry the pir gene In addition the acceptor vector which carries the ampicillin resistance gene will not be selected Therefore every colony th
37. or 1 Once you have obtained a stable cell line expressing the Tet repressor follow the steps below to transfect your stable cell line with the pe DNA4 TO E fusion vector Use Zeocin to select for double stable clones Remember to maintain your cells in medium containing blasticidin as well 2 Transfect your cell line of choice with your pecDNA4 TO E fusion vector using the desired protocol Include a sample of untransfected cells as a negative control 3 24 hours after transfection wash the cells and add fresh medium to the cells 4 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 If the cells are too dense the antibiotic will not kill the untransfected cells 5 Replenish selective medium every 3 4 days until Zeocin resistant colonies are detected 6 Pick at least 20 foci and expand them to test for tetracycline inducible gene expression Continued on next page Creation of Stable Cell Lines Continued Dual Selection of Stable Integrants Purification Preparation of Cells for Lysis Lysis of Cells If you wish to select for stable cell lines by dual selection you may cotransfect your pcDNA4 TO E fusion vector and pcDNA6 TR into your cell line of choice and select with Zeocin and blasticidin Pick and expand at least 40 foci to screen for tetracyclin
38. origin Allows rescue of single strand 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 EM 7 promoter Synthetic prokaryotic promoter for IM expression ofthe Zeocin resistance gene in E coli Zeocin resistance Sh ble gene expressed from the SV40 early promoter or the EM 7 promoter Permits selection of stable transfectants in mammalian cells and transformants in E coli Drocourt et al 1990 Mulsant et al 1988 SV40 early polyadenylation signal Allows efficient transcription termination and polyadenylation of mRNA pUC origin Permits high copy number replication and growth in E coli bla promoter Allows expression of the ampicillin bla resistance gene Ampicillin resistance gene lactamase Allows selection of transformants in E coli 19 pcDNA4 TO E Uni lacZ Description pcDNA4 TO E Uni lacZ is a 10405 bp expression control vector that contains the lacZ gene 3056 bp The lacZ gene was amplified and TOPO Cloned into pUni V5 His Gene TOPO The resulting vector was recombined with peDNA4 TO E using cre recombinase to create p DNA4 TO E Uni lacZ Note pUni V5 His Gene TOPO is similar to pUni V5 His TOPO except that it contains additional DNA between the TOPO Cloning site and the V5 epitope
39. pression in the T REx System is based on a repression derepression mechanism the amount of Tet repressor that is expressed in the host cell line from pcDNA6 TR will determine the level of transcriptional repression of the Tet operator sequences in your pe DNA4 TO E fusion vector Tet repressor levels should be sufficiently high to suitably repress basal level transcription We recommend that you cotransfect your mammalian host cell line with a ratio of at least 6 1 w w pcDNA6 TR pcDNA4 TO E fusion vector DNA You may want to try varying ratios of pcDNA6 TR pcDNA4 TO E fusion vector to optimize repression and expression for your particular cell line and your gene of interest General guidelines are provided below to cotransfect your p DNA4 TO E fusion vector or the control plasmid and pcDNA6 TR into your cell line of interest and to induce expression of your protein of interest with tetracycline Please refer to the T REx System manual for more information on transfection and the preparation and handling of tetracycline e Use cells that are approximately 60 confluent for transfection e Cotransfect your peDNA4 TO E fusion vector and peDNAG TR at a ratio of 6 1 w w into the cell line of choice using your preferred method Absolute amounts of plasmid used for transfection will vary depending on the method of transfection and the cell line used e After transfection add fresh medium and allow the cells to recover for 24
40. r competent cells please follow the manufacturer s protocol For each transformation you will need one vial of One Shot TOP10 competent cells and two selective plates Perform the following steps before beginning e Equilibrate a water bath to 42 C e Thaw the vial of SOC medium from the One Shot box and bring to room temperature e Warm LB plates containing 50 ug ml kanamycin at 37 C for 30 minutes e Thaw on ice 1 vial of One Shot cells for each transformation Continued on next page Transforming the Recombination Reaction Continued One Shot Transformation Reaction Analysis of Positive Clones 1 Add 5 ul of the recombination reaction to a vial of One Shot TOP10 E coli and mix gently Do not mix by pipetting up and down Heat shock the cells for 30 seconds at 42 C without shaking Immediately transfer the tubes to ice Add 500 ul of room temperature SOC medium Cap the tube tightly and shake the tube horizontally at 37 C for 45 minutes N BED Spread 50 ul from each transformation onto a prewarmed LB plate containing 50 ug ml kanamycin Pellet the remaining cells resuspend the cell pellet in 50 ul SOC and plate Incubate overnight at 37 C 7 An efficient recombination reaction will produce hundreds of colonies Pick 5 colonies for analysis 1 Culture the 5 colonies see previous page overnight in 2 5 ml LB or SOB medium containing 50 ug ml kanamycin See pages 16 17 for recipes for
41. s Antibiotics in this family are broad spectrum antibiotics that act as strong antibacterial and antitumor 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 CeoHsoN21021S3 and the molecular weight is 1 535 The diagram below shows the structure of Zeocin MW 1 535 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 Mammalian Cells 50 1000 ug ml varies with cell line Efficient selection requires that the concentration of NaCl be no more than 5 g liter lt 90 mM Continued on next page 21 Zeocin Continued Handli
42. t the TetO2 sequences please see below For more information about the pcDNA6 TR plasmid and the Tet repressor please refer to the T REx System manual The T REx System manual is available for downloading from our Web site www invitrogen com or from Technical Service see page 29 In the absence of tetracycline expression of your gene of interest is repressed by the binding of Tet repressor homodimers to the TetO sequences Addition of tetracycline to the cells derepresses the hybrid CMV TetO promoter in p DNA4 TO and allows expression of your gene of interest The promoters of bacterial tet genes contain two types of operator sequences O and O that serve as high affinity binding sites for the Tet repressor Hillen and Berens 1994 Hillen et al 1983 Each O and O site binds to one Tet repressor homodimer While Tet repressor homodimers bind to both tet operators with high affinity studies have shown that the affinity of the Tet repressor homodimer for O is three to five fold higher than it is for O Hillen and Berens 1994 Tet operators have been incorporated into heterologous eukaryotic promoters to allow tetracycline regulated gene expression in mammalian cells Gossen and Bujard 1992 Yao et al 1998 In the T REx System two copies of the O operator sequence TetO were inserted into the strong CMV promoter of pe DNA4 TO E to allow regulated expression of your gene of interest by tetracycline We use the
43. tion Once you have performed the recombination reaction you are ready to transform your E coli host We recommend using TOP10 E coli available with the kit for transformation but other strains are suitable E coli strains should be endonuclease A deficient endA and recombination deficient recA to ensure quality plasmid preparations and reduce the chances of recombination respectively In addition to general microbiological supplies i e plates spreaders you will need the following reagents and equipment e 42 C water bath e LB plates containing 50 ug ml kanamycin see Important below e 37 C shaking and non shaking incubator e SOC supplied in the One Shot kit It is important to select for the fusion plasmid using kanamycin Remember that the donor vector contains the R6Ky origin This origin can only be maintained in E coli strains containing the pir gene After the donor vector and p DNA4 TO E acceptor vectors have recombined to form the fusion plasmid the kanamycin resistance gene from the donor vector is linked to the pUC origin from pcDNA4 TO E The fusion plasmid can be maintained in E coli strains that do not contain the pir gene i e TOP10 By selecting for kanamycin resistance you ensure that only colonies containing the fusion vector are selected The following transformation protocol is for use with the TOP10 One Shot competent cells available with the kit If you are using othe
44. tion as is Store the stock solution at 20 C when you are finished If you wish to propagate the pcDNA4 TO E plasmid or prepare plasmid DNA you may transform the plasmid into TOP10 E coli as described on page 6 Use 10 100 ng of plasmid DNA for transformation and select transformants on LB plates containing 50 to 100 ug ml ampicillin Be sure to prepare a glycerol stock of your plasmid containing TOP10 strain for long term storage see page 9 You will need the following reagents and equipment e 100 ng of your donor vector construct e 100 ng of pcDNA4 TO E included in kit e Microcentrifuge tubes e Heat blocks set at 37 C and 65 C e Ice bucket with ice e Cre recombinase included in the kit e 10X Recombinase Buffer included in the kit 1 Set up each 20 ul recombination reaction on ice as follows Donor vector 100 ng x ul pcDNA4 TO E 100 ng y ul 10X Recombinase Buffer 2 ul Deionized water add to a total volume of 17 ul Cre Recombinase lul Final Volume 20 ul 2 Incubate at 37 C for 20 minutes 3 Incubate at 65 C for 5 minutes to inactivate the recombinase 4 Place the tube on ice and proceed to Transformation next page If you run out of time you may store the recombination reaction at 4 C or 20 C overnight Longer storage times have not been tested Transforming the Recombination Reaction Introduction Materials Supplied by the User Important Preparation for Transforma
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