pcdna 6.2/V5-pL-DEST Gateway Vector

Transcription

1 USER GUIDE pcdna 6.2/V5-pL-DEST Gateway Vector Promoterless Gateway -adapted destination vector for cloning and expression of C-terminal V5 fusion proteins in mammalian cells Catalog number Revision date 4 April 2012 Publication Part number MAN For Research Use Only. Not for human or animal therapeutic or diagnostic use.

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3 Table of Contents Important Information... iv Accessory Products... v Methods... 1 Overview... 1 Generating an Entry Clone... 3 Creating an Expression Clone... 5 Performing the LR Recombination Reaction... 7 Transfection Expression and Analysis Creating Stable Cell Lines Appendix Recipes Blasticidin Map and Features of pcdna 6.2/V5-pL-DEST Technical Support Purchaser Notification Gateway Clone Distribution Policy References iii

4 Important Information Contents The pcdna 6.2/V5-pL-DEST Gateway Vector is supplied as 60 μl of 100 ng/μl supercoiled DNA. Shipping and Storage The pcdna 6.2/V5-pL-DEST Gateway Vector is shipped at room temperature. Upon receipt, store at 20 C. Product Use For research use only. Not intended for any human or animal diagnostic or therapeutic uses. iv

5 Accessory Products Additional Products Additional products that may be used with the pcdna 6.2/V5-pL-DEST vector are available from Life Technologies. Ordering information is provided below. Product Amount Catalog No. Gateway LR Clonase II Plus Enzyme Mix MultiSite Gateway Pro 2.0 Kit for 2- fragment recombination MultiSite Gateway Pro 3.0 Kit for 3- fragment recombination MultiSite Gateway Pro 4.0 Kit for 4- fragment recombination MultiSite Gateway Pro Plus Kit for 2-, 3- or 4-fragment recombination One Shot ccdb Survival T1 R Chemically Competent E. coli One Shot Mach1 T1 R Chemically Competent E. coli PureLink HiPure Plasmid Midiprep Kit Lipofectamine reactions 100 reactions reactions reactions reactions reactions μl C μl C reactions 50 reactions 1.5 ml 0.75 ml K K Blasticidin 50 mg R Continued on next page v

6 Accessory Products, continued Detection of Recombinant Proteins You can detect expression of your recombinant fusion protein using the Anti-V5 antibodies available from Life Technologies. The amount of antibody supplied is sufficient for 25 Western blots or 25 immunostaining reactions (FITCconjugated antibody only). Product Epitope Catalog no. Anti-V5 Antibody Detects 14 amino acid epitope R Anti-V5-HRP Antibody derived from the P and V R proteins of the paramyxovirus, Anti-V5-AP Antibody SV5 (Southern et al., 1991). R Anti-V5-FITC Antibody GKPIPNPLLGLDST R vi

7 Methods Overview Description pcdna 6.2/V5-pL-DEST is a 6.7 kb vector that contains no endogenous eukaryotic promoters, and is adapted with the Gateway Technology to allow expression of one or more promoters and genes of interest of interest in a variety of mammalian systems. pcdna 6.2/V5-pL-DEST is compatible with MultiSite Gateway Pro Kits (see page v for ordering information). For more information on the Gateway Technology, see the next page. Features The pcdna 6.2/V5-pL-DEST vector contains the following elements: Two recombination sites, attr1 and attr2 for recombinational cloning of up to 4 entry clones when using MultiSite Gateway Pro kits (see page v) The ccdb gene located between the two attr sites for negative selection Chloramphenicol resistance gene located between the two attr sites for counterscreening The V5 epitope tag for detection using Anti-V5 antibodies The Herpes Simplex Virus thymidine kinase polyadenylation signal for proper termination and processing of the recombinant transcript f1 intergenic region for production of single-strand DNA in F plasmid-containing E. coli SV40 early promoter and origin for expression of the Blasticidin resistance gene and stable propagation of the plasmid in mammalian hosts expressing the SV40 large T antigen Blasticidin resistance gene for selection of stable cell lines The puc origin for high copy replication and maintenance of the plasmid in E. coli The ampicillin (bla) resistance gene for selection in E. coli Continued on next page 1

8 Overview, continued The Gateway Technology The Gateway Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (Landy, 1989) to provide a rapid and highly efficient way to move your gene of interest into multiple vector systems. To express your gene of interest using Gateway Technology, simply: 1. Generate entry clones containing your promoter and gene(s) of interest. 2. Generate an expression clone by performing an LR recombination reaction between the entry clone(s) and pcdna 6.2/V5-pL-DEST). 3. Transfect your expression clone into cells of your choice to transiently or stably express your gene of interest. For more information on the Gateway Technology and MultiSite Gateway Pro, refer to the Gateway Technology with Clonase II, and MultiSite Gateway Pro manuals. These manuals are available from or by contacting Technical Support (page 21). 2

9 Generating an Entry Clone Introduction To recombine your promoter and gene of interest into pcdna 6.2/V5-pL-DEST, you will need one or more entry clones containing the promoter and gene of interest. Refer to the manual for the system you are using (i.e., MultiSite Gateway Pro) for detailed instructions to construct entry clones. Generating Entry Clones You may generate entry clones in a number of ways: Use a MultiSite Gateway Pro Kit to create 2, 3, or 4 individual entry clones, each containing a promoter or a gene of interest (most recommended method). Use pentr /D-TOPO or pcr8/gw/topo to rapidly TOPO clone both promoter and gene of interest as a cassette. Perform a BP recombination reaction using a PCR product with flanking attb sites and an attp-containing pdonr vector to create your entry clone. For more information about these products, go to or contact Technical Support (page 21). Kozak Consensus Sequence Your gene of interest must contain a Kozak translation initiation sequence with an ATG initiation codon for proper initiation of translation (Kozak, 1987; Kozak, 1990; Kozak, 1991). An example of a Kozak consensus sequence is provided below. The ATG initiation codon is shown underlined. (G/A)NNATGN Other sequences are possible, but the G or A at position 3 (shown in bold) is critical for a functional Kozak sequence. At position +4 any of the four nucleotides can be present to form part of the Kozak sequence. Continued on next page 3

10 Generating an Entry Clone, continued Points to Consider Before Recombining pcdna 6.2/V5-pL-DEST is a C-terminal fusion vector; however, you may use the vector to express native proteins or C-terminal fusion proteins. Consider the following when generating your entry clone(s): If you wish to... Then your insert... include the V5 epitope tag should NOT contain a stop codon should be in frame with the V5 epitope tag after recombination (see page 6 for a diagram) not include the V5 epitope tag include a eukaryotic promoter should contain a stop codon should initiate with an ATG in a Kozak sequence (see previous page) 4

11 Creating an Expression Clone Introduction After you have generated an entry clone or clones, you will perform the LR recombination reaction to transfer the elements of choice into the pcdna 6.2/V5-pL-DEST vector to create your expression clone. To ensure that you obtain the best results, we recommend that you read this section and the next section entitled Performing the LR Recombination Reaction (pages 7 10) before beginning. Experimental Outline To generate an expression clone, you will: 1. Perform an LR recombination reaction using the appropriate entry clone(s) and the attr-containing pcdna 6.2/V5-pL-DEST vector. 2. Transform the reaction mixture into a suitable E. coli host. 3. Select for expression clones (refer to page 6 for a diagram of the recombination region of the resulting expression clones). Propagating the Vector If you wish to propagate and maintain pcdna 6.2/V5-pL- DEST, we recommend using One Shot ccdb Survival T1 R Chemically Competent E. coli from Life Technologies for transformation. The ccdb Survival T1 R E. coli strain is resistant to ccdb effects and can support the propagation of plasmids containing the ccdb gene. To maintain the integrity of the vector, select for transformants in media containing μg/ml ampicillin and μg/ml chloramphenicol. Note: Do not use general E. coli cloning strains including TOP10 or DH5α for propagation and maintenance as these strains are sensitive to ccdb effects. Continued on next page 5

12 Creating an Expression Clone, continued Recombination Region The recombination region of the expression clone resulting from pcdna 6.2/V5-pL-DEST a single entry clone is shown below. If multiple entry clones are used, refer to the MultiSite Gateway Pro manual for the resulting recombination region of the expression clone. Features of the Recombination Region: Shaded regions correspond to DNA sequences transferred from the entry clone into pcdna 6.2/V5- pl-dest by recombination. Non-shaded regions are derived from the pcdna 6.2/V5-pL-DEST vector. The nucleotides flanking the shaded region correspond to bases 150 and 2396 of the pcdna 6.2/V5-pL-DEST vector sequence T7 promoter/priming site CTGGCTTATC GAAATTAATA CGACTCACTA TAGGGAGACC CAAGCTGGCT AGTTAAGCTA GACCGAATAG CTTTAATTAT GCTGAGTGAT ATCCCTCTGG GTTCGACCGA TCAATTCGAT attb1 attb2 TCAACAAGTT GTACAAAAAAGC AGG CTN NAC CCA GCT TTC TTG TAC AGTTGTTCAA CATGTTTTTTCG TCC GAN --- GENE NTG GGT CGA AAG AAC ATG Pro Ala Phe Leu Tyr V5 epitope 2403 AAA GTG GTT GAT CTA GAG GGC CCG CGG TTC GAA GGT AAG CCT ATC CCT AAC TTT CAC CAA CTA GAT CTC CCG GGC GCC AAG CTT CCA TTC GGA TAG GGA TTG Lys Val Val Asp Leu Glu Gly Pro Arg Phe Glu Gly Lys Pro Ile Pro Asn V5 reverse primer binding site 2454 CCT CTC CTC GGT CTC GAT TCT ACG CGT ACC GGT TAG TAA TGA GTTTAGG GGA GAG GAG CCA GAG CTA AGA TGC GCA TGG CCA *** *** *** Pro Leu Leu Gly Leu Asp Ser Thr Arg Thr Gly Continued on next page 6

13 Performing the LR Recombination Reaction Introduction Once you have obtained an entry clone or clones containing your gene of interest, you may perform an LR recombination reaction between the entry clone(s) and pcdna 6.2/V5-pL-DEST, and transform the reaction mixture into a suitable E. coli host (see below) to select for an expression clone. We recommend including a negative control (no LR Clonase II Plus) in your experiment to help you evaluate your results. E. coli Host You may use any reca, enda E. coli strain including Mach1 T1 R, TOP10, DH5α, or equivalent for transformation (see page v for ordering information). Do not transform the LR reaction mixture into E. coli strains that contain the F episome (e.g., TOP10F ). These strains contain the ccda gene and will prevent negative selection with the ccdb gene. Continued on next page 7

14 Performing the LR Recombination Reaction, continued LR Clonase II Plus Enzyme Mix LR Clonase II Plus enzyme mix is available separately from Life Technologies and is optimized to catalyze the MultiSite Gateway Pro LR recombination reaction. The LR Clonase II Plus enzyme mix combines the proprietary enzyme formulation and 5X LR Clonase Reaction Buffer in an optimized single-tube format for easy set-up of the LR recombination reaction. Use the protocol provided on the next page to perform the LR recombination reaction using LR Clonase II Plus enzyme mix. Materials Needed You should have the following materials on hand before beginning: Purified plasmid DNA of your entry clone(s) (10 fmoles each) pcdna 6.2/V5-pL-DEST (20 fmoles) LR Clonase II Plus enzyme mix (keep at 20 C until immediately before use) 1X TE Buffer, ph 8.0 (10 mm Tris-HCl, ph 8.0, 1 mm EDTA) 2 μg/μl Proteinase K solution (supplied with the enzyme mix; thaw and keep on ice until use) Appropriate competent E. coli host and growth media for expression S.O.C. Medium LB agar plates containing 100 μg/ml ampicillin Continued on next page 8

15 Performing the LR Recombination Reaction, continued Setting Up the LR Reaction Follow this procedure to perform the LR reaction between multiple entry clones and a destination vector. To include a negative control, set up a second sample reaction, but omit the LR Clonase II Plus enzyme mix. 1. Add the following components to 1.5-mL microcentrifuge tubes at room temperature and mix. Component Entry clone (10 fmoles each) Destination vector (20 fmoles) 1X TE Buffer, ph 8.0 Sample 1 7 μl 1 μl to 8 μl 2 Remove the LR Clonase II Plus enzyme mix from 20ºC or 80ºC and thaw on ice (~2 minutes). 3 Vortex the LR Clonase II Plus enzyme mix briefly twice (2 seconds each time). 4 To each sample above, add 2 μl of LR Clonase II Plus enzyme mix. Mix well by pipetting up and down. Reminder: Return LR Clonase II Plus enzyme mix to 20ºC or 80ºC immediately after use. The enzyme mix can be stored at 20ºC for up to 6 months or at 80ºC for long term storage. 5 Incubate reactions at 25ºC for 16 hours. 6 Add 1 μl of Proteinase K solution to each reaction. Incubate for 10 minutes at 37ºC. 7 Transform 1 μl of the LR recombination reaction into One Shot Mach1 T1 R Competent Cells (follow the manufacturer s instructions) and select for expression clones. Continued on next page 9

16 Performing the LR Recombination Reaction, continued What You Should See If you use E. coli cells with a transformation efficiency of cfu/μg, a typical LR reaction should give >5,000 colonies if the entire reaction is transformed and plated. For multiple fragment reactions, typical numbers of colonies (per 10 μl LR reaction) are: 2-fragment recombination reaction: 2,000 15,000 3-fragment recombination reaction: 1,000 5,000 4-fragment recombination reaction: Confirming the Expression Clone The ccdb gene mutates at a very low frequency, resulting in a very low number of false positives. True expression clones will be ampicillin-resistant and chloramphenicol-sensitive. Transformants containing a plasmid with a mutated ccdb gene will be both ampicillin- and chloramphenicol-resistant. To check your putative expression clone, test for growth on LB plates containing 30 μg/ml chloramphenicol. A true expression clone will not grow in the presence of chloramphenicol. Sequencing To confirm that your gene of interest is in frame with the C-terminal V5 epitope, you may sequence your expression construct, if desired. We suggest using the following primer sequences. Refer to the diagram on page 6 for the location of the primer binding sites. For your convenience, Life Technologies offers a custom primer synthesis service. For more information, go to or contact Technical Support (page 21). Primer T7 Promoter V5 Reverse Sequence 5 -TAATACGACTCACTATAGGG-3 5 -ACCGAGGAGAGGGTTAGGGAT-3 10

17 Transfection Introduction This section provides general information for transfecting your expression clone into the mammalian cell line of choice. Plasmid Preparation Once you have generated your expression clone, you must isolate plasmid DNA for transfection. Plasmid DNA for transfection into eukaryotic 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 HiPure Plasmid Midiprep Kit, or CsCl gradient centrifugation. Methods of Transfection 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 available from Life Technologies (see page v). For more information about Lipofectamine 2000 and other transfection reagents, go to or contact Technical Support (page 21). Continued on next page 11

18 Expression and Analysis Introduction Expression of your gene of interest from the expression clone can be performed in either transiently transfected cells or stable cell lines (see page 14 for guidelines to create stable cell lines). You may use a functional assay or a western blot analysis to detect your recombinant protein (see below). Preparing Cell Lysates To detect your fusion protein by Western blot, you will need to prepare a cell lysate from transfected cells. A sample protocol is provided below. Other protocols are suitable. To lyse cells: 1. Wash cell monolayer (~ to cells) once with phosphate-buffered saline. 2. Scrape cells into 1 ml PBS and pellet the cells at 1500 g for 5 minutes. 3. Resuspend in 50 μl Cell Lysis Buffer (see page 17 for a recipe). Other cell lysis buffers are suitable. 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 g for 10 minutes at +4 C 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 (see page 17 for a recipe) to a final concentration of 1X and boil the sample for 5 minutes. 7. Load 20 μg of lysate onto an SDS-PAGE gel and electrophorese. Use the appropriate percentage of acrylamide to resolve your fusion protein. Continued on next page 12

19 Expression and Analysis, continued Polyacrylamide Gel Electrophoresis To facilitate separation and visualization of your recombinant fusion protein by polyacrylamide gel electrophoresis, a wide range of pre-cast NuPAGE and Novex Tris-Glycine polyacrylamide gels and electrophoresis apparatus are available from Life Technologies. For more information, go to or contact Technical Support (page 21). Detecting Recombinant Fusion Proteins To detect expression of your recombinant fusion protein by Western blot analysis, you may use the Anti-V5 antibodies available from Life Technologies (see page vi) for ordering information) or an antibody to your protein of interest. In addition, the Positope Control Protein is available from Life Technologies for use as a positive control for detection of fusion proteins containing a V5 epitope. The ready-to-use WesternBreeze Chromogenic Kits and WesternBreeze Chemiluminescent Kits are available from Life Technologies to facilitate detection of antibodies by colorimetric or chemiluminescent methods. For more information, go to or contact Technical Support (page 21). Note The C-terminal peptide containing the V5 epitope will add approximately 4 kda when fused to your protein. 13

20 Creating Stable Cell Lines Introduction The pcdna 6.2/V5-pL-DEST vector contains the Blasticidin resistance gene to allow selection of stable cell lines. If you wish to create stable cell lines, transfect your construct into the mammalian cell line of choice and select for foci using Blasticidin. General information and guidelines are provided below. RECOMMENDATION To obtain stable transfectants, we recommend that you linearize your pcdna 6.2/V5-pL-DEST 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. Blasticidin Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two blasticidin S deaminase genes: bsd from Aspergillus terreus (Kimura et al., 1994) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert blasticidin S to a nontoxic deaminohydroxy derivative (Izumi et al., 1991). Continued on next page 14

21 Creating Stable Cell Lines, continued Determining Antibiotic Sensitivity To successfully generate a stable cell line expressing your protein of interest, you need to determine the minimum concentration of Blasticidin 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. Refer to page 18 for instructions on how to prepare and store Blasticidin. 1. Plate or split a confluent plate so the cells will be approximately 25% confluent. Prepare a set of 6 7 plates. and add the following concentrations of Blasticidin to each plate: 0, 1, 3, 5, 7.5, and 10 μg/ml 2. Replenish the selective media every 3 4 days, and observe the percentage of surviving cells. 3. Count the number of viable cells at regular intervals to determine the appropriate concentration of antibiotic that prevents growth within 1 2 weeks after addition of the antibiotic. Blasticidin Selection Guidelines Once you have determined the appropriate Blasticidin concentration to use for selection, you can generate a stable cell line expressing your pcdna 6.2/V5-pL-DEST construct. Blasticidin is available separately from Life Technologies (see page v for ordering information). Use as follows: 1. Prepare a stock solution of 5 10 mg/ml of Blasticidin in sterile water. Filter-sterilize the solution. 2. Use the predetermined concentration of Blasticidin in complete medium. 3. Cells differ in their susceptibility to Blasticidin. Complete selection can take up to 10 days of growth in selective medium. Refer to page 18 for instructions on how to prepare and store Blasticidin. 15

22 Appendix Recipes LB (Luria- Bertani) Medium and Plates Composition: 1.0% Tryptone 0.5% Yeast Extract 1.0% NaCl ph 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 if needed, and pour into 10 cm plates. 4. Let harden, then invert and store at 4 C. Continued on next page 16

23 Recipes, continued Cell Lysis Buffer 50 mm Tris, ph mm NaCl 1% Nonidet P This solution can be prepared from the following common stock solutions. For 100 ml, combine 1 M Tris base 5 ml 5 M NaCl 3 ml Nonidet P-40 1 ml 2. Bring the volume up to 90 ml with deionized water and adjust the ph to 7.8 with HCl. 3. Bring the volume up to 100 ml. Store at room temperature. To prevent proteolysis, you may add 1 mm PMSF, 1 μm leupeptin, or 0.1 μm aprotinin before use. 4X SDS-PAGE Sample Buffer 1. Combine the following reagents: 0.5 M Tris-HCl, ph ml Glycerol (100%) 4 ml β-mercaptoethanol 0.8 ml Bromophenol Blue 0.04 g SDS 0.8 g 2. Bring the volume to 10 ml with sterile water. 3. Aliquot and freeze at 20 C until needed. 17

24 Blasticidin Molecular Weight, Formula, and Structure The formula for Blasticidin S is C17H26N8O5-HCl, and the molecular weight is The diagram below shows the structure of Blasticidin. NH2 N N O CH3 HOOC O -HCl H2N N NH NH NH2 O Handling Blasticidin Always wear gloves, mask, goggles, and protective clothing (e.g., a laboratory coat) when handling Blasticidin. Weigh out Blasticidin and prepare solutions in a hood. Preparing and Storing Stock Solutions Blasticidin may be obtained separately from Life Technologies (see page v) in 50 mg aliquots. Blasticidin is soluble in water. Sterile water is generally used to prepare stock solutions of 5 10 mg/ml. Dissolve Blasticidin in sterile water and filter-sterilize the solution. Aliquot in small volumes suitable for one time use (see next to last point below) and freeze at 20 C for longterm storage or store at 4 C for short-term storage. Aqueous stock solutions are stable for 1 2 weeks at 4 C and 6 8 weeks at 20 C. ph of the aqueous solution should be 7.0 to prevent inactivation of Blasticidin. Do not subject stock solutions to freeze/thaw cycles (do not store in a frost-free freezer). Upon thawing, use what you need and store the thawed stock solution at 4 C for up to 2 weeks. Medium containing Blasticidin may be stored at 4 C for up to 2 weeks. 18

25 Map and Features of pcdna 6.2/V5-pL- DEST Map The map below shows the elements of pcdna 6.2/V5-pL- DEST. DNA from the entry clone replaces the region between bases 150 and The complete sequences of these vectors are available for downloading from or by contacting Technical Support (page 21). T7 attr1 ccdb Cm R attr2 V5 epitope TK pa f1 ori SV40 ori Ampicillin pcdna 6.2/V5-pL- DEST EM7 puc ori 6693 bp Blasticidin SV40 pa Comments for pcdna 6.2/V5-pL-DEST 6693 bp T7 promoter/priming site: bp attr1 site: bp ccdb gene (c): bp Chloramphenicol resistance gene (c): bp attr2 site: bp V5 epitope: bp V5 reverse priming site: bp TK polyadenylation signal: bp f1 origin: bp SV40 early promoter and origin: bp EM7 promoter: bp Blasticidin resistance gene: bp SV40 early polyadenylation signal: bp puc origin (c): bp Ampicillin (bla) resistance gene (c): bp bla promoter (c): bp (c) = complementary strand Continued on next page 19

26 Map and Features of pcdna 6.2/V5-pL- DEST, continued Features pcdna 6.2/V5-pL-DEST (6693 bp) contains the following elements. Features have been functionally tested. Feature T7 promoter/priming site attr1 and attr2 sites ccdb gene Chloramphenicol resistance gene V5 epitope V5 reverse priming site Herpes Simplex Virus Thymidine Kinase (TK) polyadenylation signal f1 origin SV40 early promoter and origin EM7 promoter Blasticidin (bsd) resistance gene SV40 early polyadenylation signal puc origin Ampicillin (bla) resistance gene (β-lactamase) Benefit Allows in vitro transcription in the sense orientation in the presence of T7 polymerase and sequencing through the insert Allows recombinational cloning of the gene of interest from an entry clone or clones Allows negative selection of plasmid Allows counterscreening of plasmid Allows detection of the recombinant fusion protein by the Anti-V5 antibodies (Southern et al., 1991). Allows sequencing of the insert Allows efficient transcription termination and polyadenylation of mrna (Cole and Stacy, 1985) Allows rescue of single-stranded DNA Allows efficient, high-level expression of the neomycin or Blasticidin resistance gene and episomal replication in cells expressing the SV40 large T antigen Allows expression of the Blasticidin resistance gene in E. coli Allows selection of stable transfectants in mammalian cells (Kimura et al., 1994) Allows efficient transcription termination and polyadenylation of mrna Allows high-copy number replication and growth in E. coli Allows selection of transformants in E. coli 20

27 Technical Support Obtaining support For the latest services and support information for all locations, go to At the website, you can: Access worldwide telephone and fax numbers to contact Technical Support and Sales facilities Search through frequently asked questions (FAQs) Submit a question directly to Technical Support (techsupport@lifetech.com) Search for user documents, SDSs, vector maps and sequences, application notes, formulations, handbooks, certificates of analysis, citations, and other product support documents Obtain information about customer training Download software updates and patches Safety Data Sheets (SDS) Safety Data Sheets (SDSs) are available at Certificate of Analysis The Certificate of Analysis provides detailed quality control and product qualification information for each product. Certificates of Analysis are available on our website. Go to and search for the Certificate of Analysis by product lot number, which is printed on the box. Continued on next page 21

28 Technical Support, continued Limited Product Warranty Life Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at If you have any questions, please contact Life Technologies at 22

29 Purchaser Notification Introduction Use of pcdna 6.2/V5-pL-DEST Gateway Vectors is covered under the licenses detailed below. Limited Use Label License No. 358: Research Use Only The purchase of this product conveys to the purchaser the limited, non-transferable right to use the purchased amount of the product only to perform internal research for the sole benefit of the purchaser. No right to resell this product or any of its components is conveyed expressly, by implication, or by estoppel. This product is for internal research purposes only and is not for use in commercial applications of any kind, including, without limitation, quality control and commercial services such as reporting the results of purchaser s activities for a fee or other form of consideration. For information on obtaining additional rights, please contact outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California Limited Use Label License No. 54: ULB ccdb Selection Technology Notice to Purchaser This product is the subject of U.S. patents and corresponding foreign patents and is sold under license from the Université Libre de Bruxelles for research purposes only. ccdb selection technology is described in Bernard et al., "Positive Selection Vectors Using the F Plasmid ccdb Killer Gene" Gene 148 (1994) 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). For information on obtaining additional rights, please contact outlicensing@lifetech.com. 23

30 Gateway Clone Distribution Policy Introduction The information supplied in this section is intended to provide clarity concerning Life Technologies policy for the use and distribution of cloned nucleic acid fragments, including open reading frames, created using Life Technologies commercially available Gateway Technology. Gateway Entry Clones Life Technologies understands that Gateway entry clones, containing attl1 and attl2 sites, may be generated by academic and government researchers for the purpose of scientific research. Life Technologies agrees that such clones may be distributed for scientific research by non-profit organizations and by for-profit organizations without royalty payment to Life Technologies. Gateway Expression Clones Life Technologies also understands that Gateway expression clones, containing attb1 and attb2 sites, may be generated by academic and government researchers for the purpose of scientific research. Life Technologies agrees that such clones may be distributed for scientific research by academic and government organizations without royalty payment to Life Technologies. Organizations other than academia and government may also distribute such Gateway expression clones for a nominal fee ($10 per clone) payable to Life Technologies. Additional Terms and Conditions We would ask that such distributors of Gateway entry and expression clones indicate that such clones may be used only for research purposes, that such clones incorporate the Gateway Technology, and that the purchase of Gateway Clonase from Life Technologies is required for carrying out the Gateway recombinational cloning reaction. This should allow researchers to readily identify Gateway containing clones and facilitate their use of this powerful technology in their research. Use of Life Technologies Gateway Technology, including Gateway clones, for purposes other than scientific research may require a license and questions concerning such commercial use should be directed to Life Technologies licensing department at

31 References Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, 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, 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). Capone, J. P., Sharp, P. A., and RajBhandary, U. L. (1985). Amber, Ochre and Opal Suppressor trna Genes Derived from a Human Serine trna Gene. EMBO J. 4, Chen, C., and Okayama, H. (1987). High-Efficiency Transformation of Mammalian Cells by Plasmid DNA. Mol. Cell. Biol. 7, Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian Cells with DNA. Nucleic Acids Res. 15, Cole, C. N., and Stacy, T. P. (1985). Identification of Sequences in the Herpes Simplex Virus Thymidine Kinase Gene Required for Efficient Processing and Polyadenylation. Mol. Cell. Biol. 5, Felgner, P. L., Holm, M., and Chan, H. (1989). Cationic Liposome Mediated Transfection. Proc. West. Pharmacol. Soc. 32, Felgner, P. L. a., and Ringold, G. M. (1989). Cationic Liposome-Mediated Transfection. Nature 337, Izumi, M., Miyazawa, H., Kamakura, T., Yamaguchi, I., Endo, T., and Hanaoka, F. (1991). Blasticidin S-Resistance Gene (bsr): A Novel Selectable Marker for Mammalian Cells. Exp. Cell Res. 197, Kimura, M., Takatsuki, A., and Yamaguchi, I. (1994). Blasticidin S Deaminase Gene from Aspergillus terreus (BSD): A New Drug Resistance Gene for Transfection of Mammalian Cells. Biochim. Biophys. ACTA 1219, Kozak, M. (1987). An Analysis of 5 -Noncoding Sequences from 699 Vertebrate Messenger RNAs. Nucleic Acids Res. 15, Continued on next page 25

32 References, continued Kozak, M. (1991). An Analysis of Vertebrate mrna Sequences: Intimations of Translational Control. J. Cell Biology 115, Kozak, M. (1990). Downstream Secondary Structure Facilitates Recognition of Initiator Codons by Eukaryotic Ribosomes. Proc. Natl. Acad. Sci. USA 87, Landy, A. (1989). Dynamic, Structural, and Regulatory Aspects of Lambda Sitespecific Recombination. Ann. Rev. Biochem. 58, Shigekawa, K., and Dower, W. J. (1988). Electroporation of Eukaryotes and Prokaryotes: A General Approach to the Introduction of Macromolecules into Cells. BioTechniques 6, Southern, J. A., Young, D. F., Heaney, F., Baumgartner, W., and Randall, R. E. (1991). Identification of an Epitope on the P and V Proteins of Simian Virus 5 That Distinguishes Between Two Isolates with Different Biological Characteristics. J. Gen. Virol. 72, 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, Takeuchi, S., Hirayama, K., Ueda, K., Sakai, H., and Yonehara, H. (1958). Blasticidin S, A New Antibiotic. The Journal of Antibiotics, Series A 11, 1-5. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.-C., and Axel, R. (1977). Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells. Cell 11, Yamaguchi, H., Yamamoto, C., and Tanaka, N. (1965). Inhibition of Protein Synthesis by Blasticidin S. I. Studies with Cell-free Systems from Bacterial and Mammalian Cells. J. Biochem (Tokyo) 57, Life Technologies Corporation. All rights reserved. The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners. LIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE THEREOF. 26

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