user guide

For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

DES® BioEase™ Kit with pCoBlast Gateway®-adapted destination vector for the stable expression of biotinylated fusion proteins in Drosophila Schneider 2 cells

Catalog number K4140-01

Revision date 20 February 2012 Publication Part number 25-0473

MAN0000263

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Contents

Kit Contents and Storage ..................................................................................................................................... iv 

Introduction ................................................................................................................... 1 Product Overview .................................................................................................................................................. 1 

Methods ......................................................................................................................... 3 Creating an Entry Clone ........................................................................................................................................ 3 

Creating an Expression Clone .............................................................................................................................. 4 

Performing the LR Recombination Reaction ...................................................................................................... 6 

Expression and Analysis ....................................................................................................................................... 9 

Appendix ...................................................................................................................... 12 Map and Features of pMT/BioEase™-DEST ..................................................................................................... 12 

Map of pMT/BioEase™-GW/lacZ ...................................................................................................................... 14 

Accessory Products .............................................................................................................................................. 15 

Technical Support ................................................................................................................................................. 16 

Purchaser Notification ......................................................................................................................................... 17 

References .............................................................................................................................................................. 18 

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Kit Contents and Storage

Kit Contents This manual is supplied with the DES® BioEase™ Kit with pCoBlast (Cat. no. K4140-01). For more information about the Drosophila Expression System, including kit contents and their use, refer to the Drosophila Expression System manual which is also provided with this kit.

The DES® BioEase™ Kit with pCoBlast includes the following vectors. Upon receipt, store at −30°C to −10°C.

Vector Composition Amount

pMT/BioEase™-DEST 150 ng/μL in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 in a total volume of 40 μL.

6 μg

pMT/BioEase™-GW/lacZ 0.5 μg/μL in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0 in a total volume of 20 μL.

10 μg

Product Use For research use only. Not intended for any human or animal therapeutic or diagnostic use.

1

Introduction

Product Overview

Description of the System

pMT/BioEase™-DEST is a 5.4 kb vector adapted for use with the Gateway®

Technology, and is designed to allow high-level, inducible expression of biotinylated recombinant fusion proteins in Drosophila melanogaster Schneider 2 (S2) cells using the Drosophila Expression System (DES®). Biotinylated recombinant protein may then be easily detected or immobilized to a solid support for other downstream applications.

For more information about the DES® System and Gateway® Technology, see page 2.

Features of the Vector

The pMT/BioEase™-DEST vector contains the following elements:

• The Drosophila metallothionein (MT) promoter for high-level, metal-inducible expression of the gene of interest in S2 cells (Angelichio et al., 1991; Bunch et al., 1988; Maroni et al., 1986; Olsen, 1992)

• BioEase™ tag to allow biotinylation of the recombinant protein of interest for easy detection or use in other applications (see below for more information)

• Two recombination sites, attR1 and attR2, downstream of the MT promoter for recombinational cloning of the gene of interest from an entry clone

• Chloramphenicol resistance gene (CmR) located between the attR sites for counterselection

• The ccdB gene located between the attR sites for negative selection

• pUC origin for high-copy replication and maintenance of the plasmid in E. coli

• Ampicillin resistance gene for selection in E. coli

The control plasmid, pMT/BioEase™-GW/lacZ, is included for use as a positive control for transfection and expression in S2 cells.

BioEase™ Tag The BioEase™ tag is a 72 amino acid peptide derived from the C-terminus (amino acids 524–595) of the Klebsiella pneumoniae oxalacetate decarboxylase subunit (Schwarz et al., 1988). Biotin is covalently attached to the oxalacetate decarboxylase subunit and peptide sequencing has identified a single biotin binding site at lysine 561 of the protein (Schwarz et al., 1988). When fused to a heterologous protein, the BioEase™ tag is both necessary and sufficient to facilitate in vivo biotinylation of the recombinant protein of interest (see the diagram on page 5 for the location of the biotin binding site). The entire 72 amino acid domain is required for recognition by the cellular biotinylation enzymes. For more information about the cellular biotinylation enzymes and the mechanism of biotinylation, refer to the review by Chapman-Smith and Cronan, 1999.

Continued on next page

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Product Overview, Continued

The Gateway® Technology

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 in S2 cells using the Gateway® Technology, simply:

1. Clone your gene of interest into a Gateway® entry vector of choice to create an entry clone.

2. Generate an expression clone by performing an LR recombination reaction between the entry clone and a Gateway® destination vector (e.g. pMT/BioEase™-DEST).

3. Purify plasmid and co-transfect the expression clone and the selection vector (e.g. pCoBlast) into Drosophila S2 cells. Select for stable cell lines using the appropriate antibiotic.

For more detailed information about the Gateway® Technology, generating an entry clone, and performing the LR recombination reaction, refer to the Gateway® Technology manual. This manual is available from www.lifetechnologies.com/manuals or by contacting Technical Support (see page 16).

Drosophila Expression System (DES®)

The Drosophila Expression System (DES®) utilizes a cell line derived from Drosophila melanogaster, Schneider 2 (S2) cells, and a simple expression vector (e.g. pMT/BioEase™-DEST) to facilitate high-level, inducible expression of heterologous proteins. To express your recombinant protein of interest using the DES® System, you will co-transfect your pMT/BioEase™-DEST expression construct and a selection vector (e.g. pCoBlast) into S2 cells, then use antibiotic selection to select for stable transformants. Stable cell lines can be generated in 2–4 weeks. By optimizing the ratio of expression vector to selection vector, cell lines with a very high copy number of the desired gene can be generated, leading to increased expression levels of the desired protein.

For more detailed information about the DES® System, the selection vector, and generating stable cell lines, refer to the Drosophila Expression System manual. This manual is supplied with this kit, but is also available from www.lifetechnologies.com/manuals or by contacting Technical Support (see page 16).

3

Methods

Creating an Entry Clone

Introduction To recombine your DNA sequence of interest into pMT/BioEase™-DEST, you will need an entry clone containing the DNA sequence of interest. Many entry vectors are available for purchase to facilitate generation of entry clones (see table below). For more information about each vector, see www.lifetechnologies.com or contact Technical Support (see page 16).

Once you have selected an entry vector, refer to the manual for the specific entry vector you are using for instructions to construct an entry clone. All entry vector manuals are available for downloading from www.lifetechnologies.com or by contacting Technical Support (see page 16).

Entry Vector Cat. no.

pENTR™/D-TOPO® K2400-20

pENTR™/SD/D-TOPO® K2420-20

pENTR™1A 11813-011

pENTR™2B 11816-014

pENTR™3C 11817-012

pENTR™4 11818-010

pENTR™11 11819-018

Points to Consider Before Recombining into pMT/BioEase™-DEST

pMT/BioEase™-DEST allows fusion of your gene of interest to the N-terminal BioEase™ tag. Remember that your gene of interest in the entry clone must:

• Be in frame with the N-terminal BioEase™ tag after recombination

• Contain a stop codon

Note: If your insert does not contain a stop codon, several potential stop codons are provided following the attB2 site (see underlined sequences in the diagram on page 5).

Refer to page 5 for a diagram of the recombination region of pMT/BioEase™-DEST.

Creating an Expression Clone

Introduction After you have generated an entry clone, you will perform the LR recombination reaction to transfer the gene of interest into the pMT/BioEase™-DEST vector to create your expression clone. To ensure that you obtain the best possible results, we recommend that you read this section and the next section entitled Performing the LR Recombination Reaction (pages 6–8) before beginning.

Experimental Outline

To generate an expression clone, you will:

1. Perform an LR recombination reaction using the attL-containing entry clone and the attR-containing pMT/BioEase™-DEST vector. Note: Both the entry clone and the destination vector should be supercoiled (see Important Note below).

2. Transform the reaction mixture into a suitable E. coli host (see page 6).

3. Select for expression clones (see the next page for an illustration of the recombination region of expression clones in pMT/BioEase™-DEST).

The pMT/BioEase™-DEST vector is supplied as a supercoiled plasmid. Although the Gateway® Technology manual has previously recommended using a linearized destination vector for more efficient recombination, further testing has found that linearization of pMT/BioEase™-DEST is not required to obtain optimal results for any downstream application.

4

Propagating the Vector

To propagate and maintain the pMT/BioEase™-DEST vector, use the supplied150 ng/μL stock solution in TE, pH 8.0 to transform Library Efficiency® DB3.1™ Competent Cells (see page 15 for ordering). The DB3.1™ 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 50–100 μg/mL ampicillin and 15–30 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.

Entry Clone To recombine your gene of interest into pMT/BioEase™-DEST, you should have an entry clone containing your gene of interest. Refer to the Gateway® Technology manual for guidelines to choose a Gateway® entry vector and construct an entry clone. The Gateway® Technology manual is available for downloading from www.lifetechnologies.com/manuals or by contacting Technical Support (see page 16).

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Creating an Expression Clone, Continued

Recombination Region of pMT/BioEase™-DEST

The recombination region of the expression clone resulting from pMT/BioEase™-DEST × entry clone is shown below.

Features of the Recombination Region:

• Shaded regions correspond to those DNA sequences transferred from the entry clone into the pMT/BioEase™-DEST vector by recombination. Non-shaded regions are derived from the pMT/BioEase™-DEST vector.

• Bases 1135 and 2797 of the pMT/BioEase™-DEST sequence are marked.

• The biotin binding site is labeled with a *.

• Potential stop codons are underlined.

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Performing the LR Recombination Reaction

Introduction Once you have obtained an entry clone containing your gene of interest, perform an LR recombination reaction between the entry clone and the pMT/BioEase™-DEST vector, 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 entry vector) in your experiment to help you evaluate your results.

E. coli Host You may use any recA, endA E. coli strain including TOP10, DH5α™, or equivalent for transformation (see page 15 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.

Materials Needed You should have the following materials on hand before beginning:

• Purified plasmid DNA of your entry clone (50–150 ng/μL in TE, pH 8.0)

• pMT/BioEase™-DEST vector (150 ng/μL in TE, pH 8.0)

• LR Clonase® enzyme mix (keep at –80°C until immediately before use; see page 15 for ordering information)

• 5X LR Clonase® Reaction Buffer (supplied with the LR Clonase® enzyme mix)

• TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)

• 2 μg/μL Proteinase K solution (supplied with the LR Clonase® 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 to select for expression clones

Continued on next page

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Performing the LR Recombination Reaction, Continued

Setting Up the LR Recombination Reaction

1. Add the following components to 1.5 mL microcentrifuge tubes at room temperature and mix.

Component Sample Negative Control

Entry clone (100–300 ng/reaction) 1–10 μL –

Destination vector (300 ng/reaction) 2 μL 2 μL

5X LR Clonase® Reaction Buffer 4 μL 4 μL

TE Buffer, pH 8.0 to 16 μL 10 μL 2. Remove the LR Clonase® enzyme mix from –80°C and thaw on ice

(~ 2 minutes).

3. Vortex the LR Clonase® enzyme mix briefly twice (2 seconds each time).

4. To each sample above, add 4 μL of LR Clonase® enzyme mix. Mix well by pipetting up and down.

Reminder: Return LR Clonase® enzyme mix to –80°C immediately after use.

5. Incubate reactions at 25°C for 1 hour.

Note: Extending the incubation time to 18 hours typically yields more colonies.

6. Add 2 μL of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37°C.

7. Transform 1 μL of the LR recombination reaction into a suitable E. coli host (follow the manufacturer’s instructions) and select for expression clones.

Note: You may store the LR reaction at –20°C for up to 1 week before transformation, if desired.

What You Should See

If you use E. coli cells with a transformation efficiency of 1 × 108 cfu/μg, the LR reaction should give >5,000 colonies if the entire transformation is plated.

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 chloramphenicol-sensitive and ampicillin-resistant. Transformants containing a plasmid with a mutated ccdB gene will be chloramphenicol- and ampicillin-resistant. To check your putative expression clone, test for growth on LB plates containing 30 μg/mL chloramphenicol. A true expression clone should not grow in the presence of chloramphenicol.

Continued on next page

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Performing the LR Recombination Reaction, Continued

Sequencing To confirm that your gene of interest is in frame with the N-terminal tag, you may sequence your expression construct, if desired. We recommend using the following primers to help you sequence your expression construct. Refer to the diagrams on page 5 for the location of the primer binding sites in each vector.

Note: For your convenience, we offer a custom primer synthesis service. For more information, see www.lifetechnologies.com or call Technical Support (see page 16).

Primer Sequence

MT forward primer 5′-CATCTCAGTGCAACTAAA-3′

BGH reverse primer 5′-TAGAAGGCACAGTCGAGG-3′

9

Expression and Analysis

Introduction Once you have obtained purified plasmid DNA of your pMT/BioEase™-DEST expression construct, you are ready to use the DES® System to express your recombinant protein in S2 cells. You may transfect S2 cells with the pMT/BioEase™-DEST construct alone to assay for transient expression of the recombinant protein of interest or in combination with a selection vector (see the next page) to generate stable cell lines.

Plasmid Preparation

Before proceeding to transfect S2 cells, you must prepare purified plasmid DNA. Plasmid DNA for transfection into S2 cells must be very clean and free from phenol and sodium chloride. Contaminants will kill the cells, decreasing transfection efficiency. We recommend isolating DNA using the PureLink® HiPure Midiprep Kit (see page 15 for ordering information) or CsCl gradient centrifugation.

Positive Control The pMT/BioEase™-GW/lacZ plasmid is provided as a positive control for transfection and expression (see page 14 for a map) and may be used to optimize transfection conditions. The gene encoding β-galactosidase is expressed in S2 cells under the control of the metallothionein promoter. A successful transfection will result in β-galactosidase expression that can be easily assayed (see page 11).

To propagate and maintain the pMT/Biotag™/GW-lacZ plasmid:

1. Use the 0.5 μg/μL stock solution to transform a recA, endA E. coli strain like TOP10, DH5α™-T1R, or equivalent. Use 10 ng of plasmid for transformation.

2. Select transformants on LB agar plates containing 100 μg/mL ampicillin.

3. Prepare a glycerol stock of a transformant containing plasmid for long-term storage.

Continued on next page

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Expression and Analysis, Continued

Materials to Have on Hand

To express your gene of interest from the pMT/BioEase™-DEST construct using the DES® System, you will need to have the following reagents:

• Drosophila S2 cells growing in a suitable growth medium. We recommend using Schneider’s Drosophila Medium.

• Selection vector to permit generation of stable cell lines. We recommend pCoBlast.

• Selection agent to select for stable cell lines (if desired). If you use the pCoBlast selection vector, you will need Blasticidin to select for stable cell lines.

• Transfection reagent for efficient delivery of the pMT/BioEase™-DEST construct and pCoBlast (if desired) to S2 cells. We recommend using calcium phosphate (i.e. the Calcium Phosphate Transfection Kit available for purchase; see page 15) for optimal transfection efficiency.

• Copper sulfate to induce expression of your gene of interest from the pMT/BioEase™-DEST vector.

For more information about the DES® System and pCoBlast, see the Drosophila Expression System manual. This manual is supplied with Cat. no. K4140-01, but is also available for downloading from www.lifetechnologies.com/manuals or by contacting Technical Support (see page 16).

Obtaining Reagents

The DES® BioEase™ Kit with pCoBlast includes pCoBlast, Blasticidin, S2 cells, Schneider’s Drosophila Medium, and the Calcium Phosphate Transfection Kit. If desired, you may also order Blasticidin, S2 cells, medium, and transfection reagents separately (see page 15 for ordering information).

Transfecting S2 Cells and Generating Stable Cell Lines

Refer to the Drosophila Expression System manual for detailed guidelines and protocols to:

• Culture and maintain S2 cells.

• Transfect S2 cells using the reagents supplied in the Calcium Phosphate Transfection Kit.

• Co-transfect S2 cells with the pMT/BioEase™-DEST expression construct and pCoBlast, and use Blasticidin to select for stable cell lines.

• Induce expression of recombinant protein using copper sulfate.

Continued on next page

Expression and Analysis, Continued

Detecting Recombinant Protein

Because your recombinant fusion protein is biotinylated, you may take advantage of the strong association between biotin and avidin (and its analogs including streptavidin) to easily detect your recombinant protein. To detect expression of your biotinylated, recombinant fusion protein by Western blot, we recommend using the Streptavidin-HRP or the Streptavidin-AP Conjugate available for purchase (see page 15 for ordering).

The N-terminal peptide containing the BioEase™ tag, EK recognition site, and attB1 site will add approximately 9.8 kDa to your protein. Be sure to account for any additional amino acids between the fusion tag and the start of your protein.

11

Assay for β-galactosidase

If you use pMT/BioEase™-GW/lacZ as a positive control vector, you may assay for β-galactosidase expression by western blot analysis or activity assay using cell-free lysates (Miller, 1972). The β-gal Antiserum and the β-Gal Assay Kit for fast and easy detection of β-galactosidase expression is available for purchase (see page 15 for ordering).

Note: You may also detect β-galactosidase expression using Streptavidin-HRP or Streptavidin-AP conjugate (see page 15).

Purifying the Recombinant Fusion Protein

If you wish to purify your recombinant fusion protein, you may use Streptavidin-Agarose available for purchase (see page 15 for ordering). Proteins may be purified under native or denaturing conditions. For more information, refer to the Streptavidin-Agarose manual on www.lifetechnologies.com/manuals or contact Technical Support (see page 16).

Cleavage of the BioEase™ Tag

pMT/BioEase™-DEST contains an enterokinase (EK) recognition site to allow removal of the BioEase™ tag from your recombinant fusion protein, if desired. Note that after digestion with enterokinase, 11 amino acids will remain at the N-terminus of your protein (see diagram on page 5).

A recombinant preparation of the catalytic subunit of bovine enterokinase (EKMax™) is available for purchase to remove the BioEase™ tag from your recombinant fusion protein (see page 15 for ordering). Instructions for digestion are included with the product. For more information, see www.lifetechnologies.com or contact Technical Support (see page 16).

Appendix

Map and Features of pMT/BioEase™-DEST

Map of pMT/BioEase™-DEST

The map below shows the elements of pMT/BioEase™-DEST. DNA from the entry clone replaces the region between bases 1135 and 2797. The vector sequence for pMT/BioEase™-DEST is available from www.lifetechnologies.com or by contacting Technical Support (see page 16).

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Map and Features of pMT/BioEase™-DEST, Continued

Features of pMT/BioEase™-DEST

The pMT/BioEase™-DEST vector (5375 bp) contains the following elements. All features have been functionally tested.

Feature Benefit

Drosophila metallothionein (MT) promoter

Permits high-level, inducible expression of heterologous proteins (Bunch et al., 1988; Maroni et al., 1986).

MT forward priming site Allows sequencing through the insert.

BioEase™ tag Allows biotinylation of recombinant fusion protein (Schwarz et al., 1988).

Enterokinase (EK) recognition site (Asp-Asp-Asp-Asp-Lys)

Allows removal of the BioEase™ tag from your recombinant protein using an enterokinase such as EKMax™ (see page 15).

attR1 and attR2 sites Bacteriophage λ-derived DNA recombination sequences that permit recombinational cloning of the gene of interest from a Gateway® entry clone (Landy, 1989).

BGH reverse priming site Allows sequencing through the insert.

Chloramphenicol resistance gene (CmR) Allows counterselection of the plasmid.

ccdB gene Permits negative selection of the plasmid.

SV40 late polyadenylation signal Permits efficient transcription termination and polyadenylation of mRNA (Angelichio et al., 1991).

pUC origin Permits high-copy number replication and growth in E. coli.

bla promoter Allows expression of the ampicillin (bla) resistance gene.

Ampicillin (bla) resistance gene

(β-lactamase)

Allows selection of transformants in E. coli.

Map of pMT/BioEase™-GW/lacZ

Description pMT/BioEase™-GW/lacZ is a 6852 bp control vector expressing β-galactosidase, and was generated using the Gateway® LR recombination reaction between an entry clone containing the lacZ gene and pMT/BioEase™-DEST. β-galactosidase is expressed as an N-terminal fusion protein of approximately 127 kDa.

Map of pMT/BioEase™-GW/lacZ

The map below shows the elements of pMT/BioEase™-GW/lacZ. The nucleotidesequence of the vector is available from www.lifetechnologies.com or by contacting Technical Support (see page 16).

14

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15

Accessory Products

Introduction The products listed in this section are intended for use with the pMT/BioEase™-DEST Gateway® Vector. For more information, refer to www.lifetechnologies.com or call Technical Support (see page 16).

Additional Products

The products listed in this section are intended for use with the pMT/BioEase™-DEST Gateway® Vector. The DES® Blasticidin Support Kit includes pCoBlast, blasticidin, S2 cells, Schneider’s Drosophila Medium, and transfection reagents. For more information, refer to www.lifetechnologies.com or call Technical Support (see page 16).

Product Amount Cat. no.

Gateway® LR Clonase® Enzyme Mix 20 reactions 11791-019

Library Efficiency® DB3.1™ Competent E. coli 1 mL (5 × 0.2 mL) 11782-018

One Shot® TOP10 Chemically Competent E. coli

20 × 50 μL C4040-03

Schneider (S2) Cells 1 mL vial, 1 × 107 cells/mL R690-07

Schneider’s Drosophila Medium 500 mL 21720-024

Fetal Bovine Serum 500 mL 16000-044

Penicillin-Streptomycin 100 mL 15070-063

Phosphate-Buffered Saline, pH 7.4 500 mL 10010-023

PureLink® HiPure Plasmid Midiprep Kit 25 preps K2100-04

Calcium Phosphate Transfection Kit 75 reactions K2780-01

Blasticidin S HCl 50 mg R210-01

Ampicillin 5 g Q100-16

DES® Blasticidin Support Kit 1 kit K5150-01

EKMax™ 250 units E180-01

1000 units E180-02

Streptavidin-HRP Conjugate 50 μL* SA100-01

Streptavidin-AP Conjugate 125 μL* SA100-03

Streptavidin Agarose 5 mL packed bed SA100-04

β-gal Antiserum 50 μL* R901-25

β–Gal Assay Kit 1 kit K1455-01

*The amount supplied is sufficient for 25 western blots.

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Technical Support

Obtaining support

For the latest services and support information for all locations, go to www.lifetechnologies.com/support. 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 www.lifetechnologies.com/support.

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 www.lifetechnologies.com/support and search for the Certificate of Analysis by product lot number, which is printed on the box.

Limited warranty Life Technologies Corporation is committed to providing our customers with high-quality goods and services. Our goal is to ensure that every customer is 100% satisfied with our products and our service. If you should have any questions or concerns about a Life Technologies product or service, contact our Technical Support Representatives. All Life Technologies products are warranted to perform according to specifications stated on the certificate of analysis. The Company will replace, free of charge, any product that does not meet those specifications. This warranty limits the Company’s liability to only the price of the product. No warranty is granted for products beyond their listed expiration date. No warranty is applicable unless all product components are stored in accordance with instructions. The Company reserves the right to select the method(s) used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order. Life Technologies makes every effort to ensure the accuracy of its publications, but realizes that the occasional typographical or other error is inevitable. Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation. If you discover an error in any of our publications, report it to our Technical Support Representatives. Life Technologies Corporation shall have no responsibility or liability for any special, incidental, indirect or consequential loss or damage whatsoever. The above limited warranty is sole and exclusive. No other warranty is made, whether expressed or implied, including any warranty of merchantability or fitness for a particular purpose.

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Purchaser Notification

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 92008.

Limited Use Label License ULB ccdB Selection Technology

ccdB selection technology is described in Bernard et al., "Positive Selection Vectors Using the F Plasmid ccdB Killer Gene" Gene 148 (1994) 71-74. 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 licensing information for use in other than research, please contact: outlicensing@lifetech.com or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.

Limited Use Label License Drosophila Expression System (DES)

THIS PRODUCT IS LICENSED TO YOU UNDER PATENT LICENSE FROM SMITHKLINE BEECHAM CORPORATION (SB) FOR YOUR RESEARCH USE ONLY. THIS PRODUCT MAY NOT BE SOLD OR TRANSFERRED TO ANY OTHER PERSON. IN USING THIS PRODUCT, YOU AGREE TO (1) MAKE NO COMMERCIAL USE OF THIS PRODUCT; (2) INFORM INVITROGEN OF ANY INVENTIONS OR DISCOVERIES ARISING FROM YOUR USE OF THIS PRODUCT; AND (3) GRANT TO SB THE FIRST RIGHT TO NEGOTIATE A LICENSE TO SUCH INVENTIONS AND DISCOVERIES. YOU AGREE NOT TO GRANT A LICENSE TO ANY THIRD PARTY ON TERMS MORE FAVORABLE THAN THOSE OFFERED TO SB. REQUESTS FOR LICENSES FOR COMMERCIAL MANUFACTURE OR USE MAY BE QBTAINED DIRECTLY BY CONTACTING: OUTLICENSING@LIFETECH.COM OR OUT LICENSING, LIFE TECHNOLOGIES, 5791 VAN ALLEN WAY, CARLSBAD, CALIFORNIA 92008.

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References

Angelichio, P. L., Beck, J. A., Johansen, H., and Ivey-Hoyle, M. (1991). Comparison of Several Promoters and Polyadenylation Signals for Use in Heterologous Gene Expression in Cultured Drosophila Cells. Nucleic Acids Res. 19, 5037-5043.

Bunch, T. A., Grinblat, Y., and Goldstein, L. S. (1988). Characterization and Use of the Drosophila Metallothionein Promoter in Cultured Drosophila melanogaster Cells. Nucleic Acids Res. 16, 1043-1061.

Chapman-Smith, A., and J.E. Cronan, J. (1999). Molecular Biology of Biotin Attachment to Proteins. J. Nutr. 129, 477S-484S.

Landy, A. (1989). Dynamic, Structural, and Regulatory Aspects of Lambda Site-specific Recombination. Ann. Rev. Biochem. 58, 913-949.

Maroni, G., Otto, E., and Lastowski-Perry, D. (1986). Molecular and Cytogenetic Characterization of a Metallothionein Gene of Drosophila. Genetics 112, 493-504.

Miller, J. H. (1972). Experiments in Molecular Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory).

Olsen, M. K. (1992). Stable Production of an Analog of Human Tissue Plasminogen Activator from Cultured Drosophila Cells. Cytotechnology 10, 157-167.

Schwarz, E., Oesterhelt, D., Reinke, H., Beyreuther, K., and Dimroth, P. (1988). The Sodium Ion Translocating Oxalacetate Decarboxylase of Klebsiella pneumoniae. J. Biol. Chem. 263, 9640-9645.

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