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For Research Use Only. Not for human or animal therapeutic or diagnostic use.
TOPO® Tools V5 BGH pA 3′ Element Kit For rapid, directional TOPO® Joining of PCR products to an element containing an epitope tag and the bovine growth hormone polyadenylation signal
Catalog Numbers T203-20, T203-100
Revision Date 29 May 2012
Publication Part Number 25-0425
MAN0000238
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Contents
Important Information ......................................................................................................................................... v Accessory Products .............................................................................................................................................. vi
Introduction ................................................................................................................... 1 Overview ................................................................................................................................................................. 1
Methods ......................................................................................................................... 4 Using the V5 BGH pA 3′ Element ........................................................................................................................ 4 Transfection Guidelines ........................................................................................................................................ 6
Appendix ...................................................................................................................... 14 Recipes ................................................................................................................................................................... 14 Technical Support ................................................................................................................................................ 17 Purchaser Notification ........................................................................................................................................ 18 References ............................................................................................................................................................. 19
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Important Information
Shipping/Storage The TOPO® Tools V5 BGH pA 3′ Element Kit is shipped on dry ice. Upon receipt, store the TOPO®-adapted element at –80°C. Store the rest of the kit at –20°C.
Types of Kits This manual is supplied with the following kits.
Kit Reactions Catalog No. V5 BGH pA 3′ Element 20 T203-20
100 T203-100
Kit Components The components provided in the TOPO® Tools V5 BGH pA 3′ Element Kit are
listed below. Store the TOPO®-adapted element at –80°C and the rest of the kit at –20°C.
Item Concentration Amount/
20 Rxn Kit Amount/
100 Rxn Kit V5 BGH pA 3′ Element, TOPO®-adapted
38 ng/μL double-strand DNA in: 15.63 mM Tris-HCl, pH 8.0 3.85% glycerol (w/v) 0.78 mM EDTA 0.63 mM dithiothreitol (DTT) 0.04% Triton X-100 (v/v)
20 μL 100 μL
BGH Reverse Primer #2 Lyophilized in TE Buffer, pH 8.0 3 μg 15 μg
250 mM Tris-HCl, pH 7.5 250 mM Tris-HCl, pH 7.5 250 μL CAT Control PCR Template 10 ng/μL in TE Buffer, pH 8.0 10 μL
CAT Control PCR Primers: 5′ CAT Control and 3′ CAT Control Primer
100 ng/μL each in TE Buffer, pH 8.0
10 μL
PCR Primers The table below provides the sequence and pmoles of the BGH Reverse Primer
#2 and the CAT control primers.
Primer Sequence Amount/ 20 Rxn Kit
Amount/ 100 Rxn Kit
BGH Reverse #2
5´-AAGCCATAGAGCCCACCGCA-3´ 496 pmoles 2.48 nmoles
5′ CAT Control
5′-CGGAACAAGGGACCATGGAGAAAAAAATCACTGGATA-3′ 87 pmoles
3′ CAT Control
5′-TGAGTCAAGGGCGCCCCGCCCTGCCACTCATCG-3′ 100 pmoles
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Accessory Products
Additional Products
The table below lists reagents including TOPO® Tools 5′ Element Kits that may be used with the TOPO® Tools V5 BGH pA 3′ Element Kit. Ordering information for these reagents is provided below. Note: Many other TOPO® Tools products are available from Life Technologies. For more information, please see our website (www.lifetechnologies.com) or contact Technical Support (see page 17).
Item Amount Catalog No. BGH Reverse Primer #2 3 μg N627-03
TOPO® Tools PCMV 5′ Element Kit 20 reactions T101-20
100 reactions T101-100
TOPO® Tools PCMV/TetO 5′ Element Kit 20 reactions T102-20
100 reactions T102-100 pUB/Bsd TOPO® Cloning Kit 20 reactions K512-20 PLATINUM® Taq DNA Polymerase High Fidelity
100 units 11304-011
500 units 11304-029 ThermalAce™ DNA Polymerase 200 units E0200 1000 units E1000 Lipofectamine® 2000 Reagent 0.75 mL 11668-027 1.5 mL 11668-019 S.N.A.P.™ MiniPrep Kit 25 reactions K1900-25 100 reactions K1900-01 CAT Antiserum* 50 μL R902-25
*The amount supplied is sufficient to perform 25 western blots using 10 mL working solution per reaction
Detection of Recombinant Proteins
If you TOPO® Join your PCR product in frame with the V5 epitope tag contained in the V5 BGH pA 3′ element, you may detect expression of your recombinant fusion protein using the Anti-V5 antibodies available from Life Technologies (see below). The amount of antibody supplied is sufficient for 25 western blots.
Product Epitope Catalog No.
Anti-V5 Antibody Detects 14 amino acid epitope derived from the P and V proteins of the paramyxovirus, SV5 (Southern et al., 1991) GKPIPNPLLGLDST
R960-25 Anti-V5-HRP Antibody R961-25 Anti-V5-AP Antibody R962-25
Product Use For Research Use Only. Not for human or animal therapeutic or diagnostic use.
1
Introduction
Overview
Introduction The TOPO® Tools V5 BGH pA 3′ Element Kit allows rapid topoisomerase I-mediated joining (“TOPO® Joining”) of a PCR product of interest to an element containing the V5 epitope tag and the bovine growth hormone (BGH) polyadenylation signal using TOPO® Tools technology. Concurrent TOPO® Joining of your PCR product to a TOPO® Tools 5′ element containing a promoter of choice allows creation of a linear DNA template. Amplification of this linear DNA template produces a linear DNA construct which can be used directly in mammalian transfection and expression experiments.
TOPO® Tools Technology
TOPO® Tools technology utilizes the unique properties of vaccinia DNA topoisomerase I to facilitate rapid, directional TOPO® Joining of PCR products to a choice of 5′ and 3′ elements. Depending on the 5′ and 3′ elements chosen, the same PCR product can be used in a choice of different gene analysis applications including expression studies, RNA probe generation, and protein function determination. For a detailed discussion of the TOPO® Tools technology and how TOPO® Joining works, please refer to the TOPO® Tools Technology manual. A wide variety of TOPO® Tools products are available. For more information about the TOPO® Tools products available and their applications, please refer to our website (www.lifetechnologies.com) or call Technical Support (see page 17).
The V5 BGH pA 3′ Element
The TOPO® Tools V5 BGH pA 3′ element consists of the following:
• A tag containing the V5 epitope (Southern et al., 1991) for detection of your recombinant protein with the Anti-V5 antibodies
• The BGH polyadenylation signal for efficient transcription termination and polyadenylation of mRNAs (Goodwin and Rottman, 1992)
Manuals Supplied The TOPO® Tools V5 BGH pA 3′ Element Kit is available with the following
two manuals: 1. TOPO® Tools V5 BGH pA 3′ Element Kit manual 2. TOPO® Tools Technology manual The content of each manual differs (see the next page), but the two are designed to be used together in your TOPO® Tools application. Before beginning the TOPO® Tools procedure, we recommend reading through each manual to familiarize yourself with the procedure.
Continued on next page
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Overview, continued
What This Manual Covers
This manual provides the following information:
• Sequence and other specific information pertaining to the use of the V5 BGH pA 3′ element
• General guidelines to transfect your linear DNA construct into mammalian cells
• General guidelines to detect expression of your protein of interest
What the TOPO® Tools Technology Manual Covers
The TOPO® Tools Technology manual is available with the TOPO® Tools V5 BGH pA 3′ Element Kit. The manual provides: • An overview of the TOPO® Tools technology
• Instructions to perform the TOPO® Tools procedure (see below) to generate a linear DNA construct
• Sequence information for the CAT control PCR template • Instructions to perform the control reaction using the CAT control PCR
template The TOPO® Tools Technology manual may also be downloaded from our website (www.lifetechnologies.com) or requested from Technical Support (see page 17).
Performing the TOPO® Tools Procedure
Performing the TOPO® Tools procedure involves the following steps: 1. Designing PCR primers to amplify your gene of interest
2. TOPO® Joining your PCR product to the V5 BGH pA 3′ element and an appropriate TOPO® Tools 5′ element to create a linear DNA template
3. Amplifying the linear DNA template to generate a linear DNA construct You will need to use the sequence information provided in this manual to help you design PCR primers to amplify and TOPO® Join your gene of interest to the V5 BGH pA 3′ Element.
High Throughput Applications
The TOPO® Tools V5 BGH pA 3′ Element Kit may be used to produce linear DNA constructs for mammalian transfection and expression in a high-throughput (HTP) format. Guidelines to perform the TOPO® Tools procedure in HTP format are provided in the TOPO® Tools Technology manual. General guidelines to transfect mammalian cells in an HTP format are provided on pages 9–10 of this manual.
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Experimental Outline
Experimental Outline
The table below describes the major steps required to generate a linear DNA construct containing your PCR product for use in mammalian transfection and expression experiments. For each step, please refer to the relevant manual as listed.
Step Action Manual
1 Design PCR primers to amplify your gene of interest for TOPO® Joining to the V5 BGH pA 3′ Element and an appropriate TOPO® Tools 5′ element. Consult the diagram on page 5 to help you design your reverse PCR primer. Note: Consult the diagram in your TOPO® Tools 5′ element manual to help you design your forward PCR primer.
This manual, pages 4–5 and the TOPO® Tools Technology manual
2 Amplify your gene of interest with a thermostable DNA polymerase.
TOPO® Tools Technology manual
3 TOPO® Join your PCR product to the V5 BGH pA 3′ element and an appropriate TOPO® Tools 5′ element to create a linear DNA template.
4 Amplify your linear DNA template with a thermostable DNA polymerase to generate a linear DNA construct.
5 Use agarose gel electrophoresis to verify the integrity and estimate the concentration of your linear DNA construct.
6 Purify the linear DNA construct using the S.N.A.P.™ MiniPrep Kit.
This manual, pages 7 and 16
7 Transfect the linear DNA construct into your mammalian cell line of choice.
This manual, pages 6–8
8 Assay for expression of your gene of interest. This manual, pages 11–13
4
Methods
Using the V5 BGH pA 3′ Element
Introduction Before using the V5 BGH pA 3′ element for TOPO® Joining, you will first need to design appropriate PCR primers to amplify your gene of interest. In order for TOPO® Joining to the V5 BGH pA 3′ element (and to the TOPO® Tools 5′ element) to occur, your PCR product must contain specific sequences on the 5′ and 3′ ends. By properly designing your PCR primers, you can incorporate these sequences into your PCR product. General guidelines and a diagram are provided in this section to help you design your reverse PCR primer. Specific instructions to design PCR primers and produce your PCR product, perform TOPO® Joining, and amplify the linear DNA template are provided in the TOPO® Tools Technology manual.
Features of the V5 BGH pA 3′ Element
The V5 BGH pA 3′ Element contains the following features:
• Supplied as a linear, double-strand element • Adapted with topoisomerase I on the 5′ end at the topoisomerase I
recognition site (CCCTT)
• Contains a six base pair single-strand overhang at 5′ end (GACTCA) which is complementary to the first six base pairs on the 3′ end of your PCR product
General Guidelines to Design Reverse PCR Primer
Proper design of the PCR primers used to amplify your gene of interest is critical to the success of the TOPO® Tools procedure. When designing your reverse PCR primer, please consider the following points below. For guidelines to design your forward PCR primer, please refer to the manual for the TOPO® Tools 5′ element you are using.
• Make sure that you include the 11 base pair sequence (5′-TGAGTCAAGGG-3′) necessary to facilitate directional TOPO® Joining on the 5′ end of the primer. For more information, see the TOPO® Tools Technology manual.
• If you want your PCR product to include the V5 epitope tag, design the reverse primer so that your open reading frame (ORF) is in frame with the tag and does not contain a stop codon. Note: Designing your reverse primer to remove the native stop codon and add the 11 base pair sequence (see above) will result in generation of a PCR product that will automatically be in frame with the V5 epitope tag when TOPO® Joined with the V5 BGH pA 3′ element.
• If you do not want your PCR product to include the V5 epitope tag, design your reverse primer to include a stop codon or to anneal downstream of the native stop codon.
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Using the V5 BGH pA 3′ Element, continued
Note If you do not TOPO® Join your PCR product in frame with the V5 epitope tag, then you will need to do one of the following to detect your recombinant protein:
• Have an antibody to your protein of interest • Have a functional assay to detect your protein
Sequence of the V5 BGH pA 3′ Element
To facilitate TOPO® Joining with your PCR product, the V5 BGH pA 3′ element is supplied as a linear fragment adapted with topoisomerase I on the 5′ end. The diagram below shows the sequence of the V5 BGH pA 3′ element including the TOPO® Joining site. The numbering corresponds to the size of the V5 BGH pA 3′ element before TOPO® Joining with the PCR product. The complete sequence of the V5 BGH pA 3′ element may also be downloaded from our website (www.lifetechnologies.com) or requested from Technical Support (see page 17).
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Transfection Guidelines
Introduction Before using the linear DNA construct to transfect your mammalian cell line of interest, you should have already performed the following steps: 1. TOPO® Joined your PCR product to the V5 BGH pA 3′ element and an
appropriate TOPO® Tools 5′ element to generate a linear DNA template 2. Amplified the linear DNA template to produce the linear DNA construct 3. Confirmed the integrity and estimated the concentration of the linear DNA
construct For more information about any of these steps, please refer to the TOPO® Tools Technology manual. General guidelines and recommendations to transfect the linear DNA construct into mammalian cells are provided in this section. Please note that transfection conditions and protocols will vary depending on the cell type, growth conditions, and transfection reagent that you are using. We recommend including a positive control in your experiment to help you evaluate your transfection and expression results (see below). If you are performing transfection experiments in an HTP format, please read through this section and refer to pages 9–10 for specific recommendations.
Positive Control The CAT control PCR template encodes the chloramphenicol acetyltransferase
(CAT) gene and is provided in the kit for use as a control for TOPO® Joining to a TOPO® Tools 5′ and 3′ element. Once the CAT control construct is generated (refer to the TOPO® Tools Technology manual for instructions), you may use the linear DNA construct as a positive control for mammalian transfection and expression. A successful transfection will result in CAT expression that can be easily assayed (see page 13).
Factors Affecting Transfection Efficiency
Many factors may affect the success of your transfection experiment. Please consider the following factors when designing your transfection experiment:
• Concentration and amount of linear DNA construct to transfect • Purity and quality of the linear DNA construct • Cell type to transfect
• The number of cells to transfect • Method of transfection and type of transfection reagent to use Each factor is discussed in more detail on the following pages.
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Transfection Guidelines, continued
How Much Linear DNA Construct to Use
For a 50 μL PCR reaction, we typically obtain yields in the range of 2–6 μg for a linear DNA construct of 1.3–4 kb. The DNA concentration generally ranges from 50–125 ng/μL of which we use 5–20 μL per transfection. In general, we recommend using 0.2–1 μg of linear DNA construct for each transfection (see the next page for cell number and well-size). If your yield or concentration of linear DNA construct is low: • You may purify and concentrate the linear DNA construct before use. We
generally use a resin-based procedure to purify and concentrate linear DNA constructs. A protocol to purify and concentrate linear DNA constructs using the S.N.A.P.™ MiniPrep Kit available from Life Technologies is provided in the Appendix, page 16.
• You may perform multiple secondary PCR reactions and pool the PCR reactions to generate sufficient linear DNA construct for your needs.
Purity of the Linear DNA Construct
To achieve successful transfection, your linear DNA construct must be of sufficient quality and purity. Contaminants in the PCR reaction may kill the cells or interfere with lipids, decreasing transfection efficiency. We recommend purifying the linear DNA construct before transfection. To purify the linear DNA construct, we use the protocol provided in the Appendix, page 16. Other protocols are suitable. Note: Depending on the DNA polymerase that you used to generate your linear DNA construct, the cell type you are transfecting, and the transfection reagent you are using, there may be cases where purification of the linear DNA construct is not required. Some empirical experimentation may be required to determine your optimal transfection conditions.
Cell Types to Transfect
You may use any mammalian cell line of choice for your transfection experiments. We have successfully transfected and expressed TOPO® Tools linear DNA constructs in both adherent and suspension cells. If you do not have a prerequisite cell line of choice, we recommend using the following cell lines listed below for your transfection experiments. The cell lines may be obtained from the American Type Culture Collection (ATCC). The ATCC number for each cell line is included. For more information about these cell lines, please refer to the ATCC website (www.atcc.org).
Cell Type Recommended Cell Line ATCC Number
Adherent HEK-293 (Graham et al., 1977) CRL-1573 Chinese Hamster ovary (CHO-K1)
(Kao and Puck, 1968) CCL-61
Suspension CHO-K1 (Kao and Puck, 1968) CCL-61
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Transfection Guidelines, continued
For HTP applications, we recommend using CHO-K1 suspension cells. If you have adherent CHO-K1 cells and wish to perform transfection and expression experiments using suspension cells, note that adherent CHO-K1 cells may be easily adapted to suspension culture using CHO-S-SFM II medium from Life Technologies (Catalog No. 12052-114). For more information, please see our website (www.lifetechnologies.com) or call Technical Support (see page 17).
How Many Cells to Transfect
Because the amount of linear DNA construct generated from PCR amplification is small compared to traditional plasmid preparation methods, we recommend performing transfection experiments in 6-well or 96-well plates. When using 293 or CHO-K1 adherent cells (see above), we generally plate 1 × 105 cells in 6-well or 96-well plates one day prior to transfection. When transfecting CHO-K1 suspension cells, 1 × 105 cells are added to a 96-well plate containing the DNA and transfection reagent mixture. Note that cell numbers may vary depending on the growth characteristics of your particular cell line.
Method of Transfection
To obtain high transfection efficiencies across a wide variety of cell lines and for general ease-of-use, we recommend using lipid-based reagents to transfect your mammalian cells. We typically use Lipofectamine® 2000 Reagent available from Life Technologies (see page vi for ordering information) to transfect either adherent (HEK-293 or CHO-K1) or suspension (CHO-K1) cells. Other lipid-based transfection reagents are also suitable.
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High-Throughput Transfection
Introduction For HTP applications, you may use linear DNA constructs amplified in a 96-well plate directly in transfection and expression experiments. We generally purify the linear DNA constructs in 96-well format before transfection. However, purification may not be necessary depending on the DNA polymerase that you used to produce the linear DNA construct and the transfection reagent you are using. Some empirical experimentation may be required to determine your optimal transfection conditions.
Purifying PCR Products in 96-well Format
We use the QIAvac 96 vacuum manifold and the QIAquick 96 PCR Purification Kit from Qiagen to purify linear DNA constructs in a 96-well format. Other kits or methods are suitable.
Note You may use any mammalian cell line and your protocol of choice to transfect the
linear DNA constructs in a 96-well format. Please refer to the manufacturer of your transfection reagent for instructions to transfect cells. For transfection studies in a 96-well format, we use:
• Suspension CHO-K1 cells (1 × 105 cells/well). • Lipofectamine® 2000 Reagent. Lipofectamine® 2000 may be added directly to
cells in culture medium in the presence of serum, if required. Removal of the lipid:DNA complexes and media changes are not required following transfection.
A protocol to transfect suspension CHO-K1 cells using Lipofectamine® 2000 is provided on the next page.
The transfection protocol on the next page is performed in a volume of approximately 1.1 mL. If you are using this protocol, make sure that you use 96-well plates that hold a volume of at least 1.5 mL. We generally use 96-well Flat Bottom Blocks (Qiagen, Valencia, CA; Catalog No. 19579) for our transfection experiments. The 96-well flat bottom blocks hold a volume of 2 mL.
Materials Needed If you are using Lipofectamine® 2000 Reagent to transfect suspension CHO-K1
cells, make sure that you have the following reagents on hand before beginning: • Lipofectamine® 2000 Reagent (Catalog No. 11668-027)
• OPTI-MEM® I Reduced-Serum Medium (Catalog No. 31985-070) • CHO-S-SFM II Medium (Catalog No. 12052-114) • 96-well Flat Bottom Blocks (Qiagen, see above) • Linear DNA constructs of interest in 96-well plate (after secondary
amplification; see TOPO® Tools Technology manual)
Continued on next page
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High-Throughput Transfection, continued
Transfecting Suspension CHO-K1 Cells Using Lipofectamine® 2000
Follow the protocol below to transfect suspension CHO-K1 cells using Lipofectamine® 2000 Reagent. For more details about Lipofectamine® 2000, refer to the Lipofectamine® 2000 manual. The Lipofectamine® 2000 manual is available for downloading from our website or by calling Technical Support (see page 17). 1. One day prior to transfection, resuspend suspension CHO-K1 cells in fresh
CHO-S-SFM II at a concentration of 1 × 105 cells/mL. Note: Do not resuspend the cells in 96-well plates.
2. On the day of transfection, aliquot 50 μL of OPTI-MEM® into each well of a 96-well flat bottom block.
3. Transfer 5–20 μL (50–125 ng/μL) of the linear DNA construct directly from each well of a 96-well plate into the 96-well flat bottom block containing the OPTI-MEM®.
4. In a microcentrifuge tube, dilute the Lipofectamine® 2000 into OPTI-MEM®. For each well of cells, mix together 2–5 μL of Lipofectamine® 2000 with 50 μL of OPTI-MEM®. Adjust the volumes of each component accordingly for multiple wells. Incubate the mixture for 5 minutes at room temperature. Note: Do not incubate the mixture for longer than 30 minutes.
5. Add 50 μL of the diluted Lipofectamine® 2000 mixture to each well containing the linear DNA construct.
6. Mix gently and incubate at room temperature for 20 minutes to allow the DNA:Lipofectamine® 2000 complexes to form.
7. While the DNA:Lipofectamine® 2000 mixture is incubating, prepare the suspension CHO-K1 cells by spinning the cells for 5 minutes at 1000 × g to pellet. Resuspend the cells in fresh CHO-S-SFM II without antibiotics at a concentration of 1 × 105 cells/mL.
8. Add 1 mL of suspension CHO-K1 cells to each well containing the DNA:Lipofectamine® 2000 mixture. Mix gently by rocking the plate back and forth.
9. Incubate for 24 hours at 37°C in a CO2 incubator. Proceed to assay for expression of your protein(s).
11
Expression Guidelines
Introduction Guidelines to harvest cells and assay for expression of your protein of interest are provided in this section.
Assaying for Expression
We generally harvest cells and assay for expression of the protein of interest 24 hours posttransfection, but this may vary depending on the nature of your protein and the transfection reagent used. Some experimentation may be necessary to determine the optimal expression conditions for your protein of interest.
Materials Needed We recommend using the protocols below and on the next page to prepare cell
lysates for Western blot analysis. Other protocols are suitable. Before beginning, you should have the following materials on hand:
• 1X DNase/PMSF (see recipe, page 14) • 4X SDS-PAGE Sample Buffer (see recipe, page 14)
• Phosphate buffered saline (PBS; if harvesting adherent cells; see recipe, page 15)
• Cell scraper (if harvesting adherent cells) • Microcentrifuge tubes
• Boiling water bath • Pasteur pipettes (if harvesting suspension cells) • 96-well PCR plate and thermocycler (if harvesting suspension cells in 96-well
format)
Harvesting Adherent Cells
Use the protocol below to harvest adherent cells and prepare lysates for Western blot analysis. 1. Remove medium and wash cells once with phosphate-buffered saline (PBS). 2. Add 30 μL of 1X DNase/PMSF and scrape cells. 3. Transfer the lysate to a microcentrifuge tube.
4. Add 10 μL of 4X SDS-PAGE Sample Buffer. 5. Boil sample(s) for 10 minutes.
6. Load 20 μL of the cell lysate on a polyacrylamide gel (see the next page) and electrophorese. Use the appropriate percentage of acrylamide to resolve your protein.
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Expression Guidelines, continued
Harvesting Suspension Cells
Use the protocol below to harvest suspension cells and prepare lysates for Western blot analysis. 1. Transfer the cells to a conical tube and centrifuge at 1000 × g for 5 minutes at
+4°C to pellet. 2. Remove the supernatant with a Pasteur pipette.
3. Add 30 μL of 1X DNase/PMSF. Pipet up and down to mix. 4. Freeze samples in dry ice. 5. Thaw samples at room temperature.
6. Add 10 μL of 4X SDS-PAGE Sample Buffer to the lysate. 7. Boil samples for 5 minutes.
8. Load 20 μL of each cell lysate on a polyacrylamide gel (see below) and electrophorese. Use the appropriate percentage of acrylamide to resolve your protein.
Harvesting Suspension Cells in a 96-well Format
Use the protocol below to harvest suspension cells in a 96-well format and prepare lysates for Western blot analysis. 1. Centrifuge cells in the 96-well plate at 1000 × g for 5 minutes at +4°C to pellet. 2. Remove the supernatant with a Pasteur pipette.
3. Add 30 μL of 1X DNase/PMSF to each well. 4. Freeze samples in dry ice. 5. Thaw samples at room temperature.
6. Transfer the cell lysates to a 96-well plate containing 10 μL of 4X SDS-PAGE Sample Buffer.
7. Heat samples for 5 minutes in a thermocycler.
8. Load 20 μL of each cell lysate on a polyacrylamide gel (see below) and electrophorese. Use the appropriate percentage of acrylamide to resolve your protein.
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. The NuPAGE® Gel System* avoids the protein modifications associated with Laemmli-type SDS-PAGE, ensuring optimal separation for protein analysis. In addition, Life Technologies also carries a large selection of molecular weight protein standards and staining kits. For more information about the appropriate gels, standards, and stains to use to visualize your recombinant protein, please refer to our website (www.lifetechnologies.com) or call Technical Support (see page 17).
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Expression Guidelines, continued
Detecting Expression
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 (Catalog No. R900-50) 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, please refer to our website (www.lifetechnologies.com) or call Technical Support (see page 17).
Note If you have TOPO® Joined your gene of interest in frame with the V5 epitope tag,
note that the epitope tag and additional downstream amino acids will add approximately 3 kDa to the size of your protein (see page 5 for a diagram).
Assay for CAT Protein
The CAT protein expressed from the control expression cassette is linked in frame with the V5 epitope tag and is approximately 29 kDa in size. You may assay for CAT expression by ELISA assay, western blot analysis, fluorometric assay, or radioactive assay (Ausubel et al., 1994; Neumann et al., 1987). For western blot analysis, you may use the Anti-V5 antibodies or CAT Antiserum available from Life Technologies (see page vi for ordering information). Other commercial kits that assay for CAT protein are also available from Roche Molecular Biochemicals (Catalog No. 1 363 727) and Molecular Probes (Catalog No. F-2900).
Remember that expression of your recombinant protein of interest from the linear DNA construct is transient. There is no selection marker to allow generation of stable cell lines. If you wish to create an expression vector containing your linear DNA construct, please see below.
Creating an Expression Vector
If you wish to keep an “archival copy” of your linear DNA construct, the pUB/Bsd TOPO® Cloning Kit is available from Life Technologies (see page vi for ordering information). The kit contains the pUB/Bsd-TOPO® vector to allow cloning of PCR products into an expression vector suitable for generation of stable cell lines. The pUB/Bsd-TOPO® vector contains the following features: • Topoisomerase I-activated vector for rapid cloning of PCR products
• Blasticidin resistance gene (bsd) for selection in mammalian cells • Ampicillin resistance gene for selection in E. coli • pUC origin of replication for high-copy replication and maintenance of the
plasmid in E. coli For more information, please refer to our website (www.lifetechnologies.com) or call Technical Support (see page 17).
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Appendix
Recipes
10X DNase 1 mg/mL DNase I (grade II, Roche Molecular Biochemicals, Catalog No. 104 159) 50 mM MgCl2 334 mM Tris-HCl, pH 7.5 50% glycerol 1. Prepare 1 M MgCl2 and 1 M Tris-HCl, pH 7.5 solutions. 2. Add 100 mg of DNase I (grade II) to a sterile beaker containing the reagents
listed below. Mix well. 1 M MgCl2 5 mL 1 M Tris-HCl, pH 7.5 33.4 mL Glycerol 50 mL
3. Bring up the volume to 100 mL with sterile water. 4. Aliquot and store at +4°C.
200 mM PMSF Phenylmethylsulfonyl fluoride (PMSF)
Isopropanol 1. To prepare a 200 mM stock solution, dissolve 34.8 mg in 1 mL of isopropanol. 2. Store at –20°C wrapped in foil.
1X DNase/PMSF 1X DNase
2 mM PMSF 1. For 1 mL, mix together the following reagents:
10X DNase (see recipe above) 100 μL 200 mM PMSF (see recipe above) 10 μL
2. Bring up the volume to 1 mL with sterile water. 3. Use immediately.
4X SDS-PAGE Sample Buffer
1. Combine the following reagents: 0.5 M Tris-HCl, pH 6.8 5.0 mL Glycerol (100%) 4.0 mL β-mercaptoethanol 0.8 mL Bromophenol blue 0.04 g SDS 0.8 g
2. Bring up the volume to 10 mL with sterile water. 3. Aliquot and freeze at –20°C until needed.
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Recipes, continued
Phosphate-Buffered Saline (PBS)
137 mM NaCl 2.7 mM KCl 10 mM Na2HPO4 1.8 mM KH2PO4
1. Dissolve: 8 g NaCl 0.2 g KCl 1.44 g Na2HPO4 0.24 g KH2PO4
in 800 mL deionized water. 2. Adjust pH to 7.4 with concentrated HCl. 3. Bring the volume to 1 liter. You may wish to filter-sterilize or autoclave the
solution to increase shelf life.
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Using the S.N.A.P.™ MiniPrep Kit to Purify the Linear DNA Construct
Introduction We generally purify the linear DNA construct prior to transfection to obtain
optimal transfection efficiencies. You may use the S.N.A.P.™ MiniPrep Kit (Catalog No. K1900-25) and the protocol below to purify your linear DNA construct. If your linear DNA construct has not amplified efficiently and the yield of your PCR product is low, you may also use the S.N.A.P.™ MiniPrep Kit to concentrate the linear DNA construct before transfection.
Materials Needed You should have the following reagents on hand before beginning:
• Isopropanol • Binding Buffer (supplied with the S.N.A.P.™ MiniPrep Kit )
• Wash Buffer (supplied with the S.N.A.P.™ MiniPrep Kit) • Final Wash Buffer (supplied with the S.N.A.P.™ MiniPrep Kit) • Sterile water • S.N.A.P.™ MiniPrep columns (supplied with the S.N.A.P.™ MiniPrep Kit)
Purification Protocol
Follow the protocol below to purify your linear DNA construct using the S.N.A.P.™ MiniPrep Kit. The protocol provides instructions to purify PCR products from a 50 μL reaction volume. To purify PCR products from larger reaction volumes (e.g. several PCR reactions pooled together), scale up the volumes of each buffer accordingly. Note: Details about the components of the S.N.A.P.™ MiniPrep Kit may be found in the S.N.A.P.™ MiniPrep Kit manual. The manual is available for downloading from our website (www.lifetechnologies.com) or by calling Technical Support (see page 17).
1. Mix the 50 μL amplification reaction with 150 μL of Binding Buffer. Mix well.
2. Add 350 μL of isopropanol. Mix well. 3. Load solution from Step 2 onto a S.N.A.P.™ MiniPrep column. Centrifuge for
30 seconds at 1000 × g in a microcentrifuge and discard the flow-through.
4. Add 250 μL of the Wash Buffer and centrifuge for 30 seconds at 1000 × g in a microcentrifuge. Discard the flow-through.
5. Add 450 μL of the Final Wash Buffer and centrifuge for 30 seconds at 1000 × g in a microcentrifuge. Discard the flow-through.
6. Centrifuge for an additional 10 seconds at full-speed in a microcentrifuge to dry the column.
7. Transfer the column to a new collection tube. Add 30 μL of sterile water to the column. Incubate at room temperature for 1 minute. Note: If you are purifying PCR products from pooled PCR reactions, do not increase the volume of sterile water that you use to elute the DNA.
8. Centrifuge for 30 seconds at full-speed in a microcentrifuge to elute the DNA. Collect the flow-through. Use 5–20 μL (50–125 ng/μL) for each transfection.
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Technical Support
Obtaining Support For the latest services and support information for all locations, go to www.lifetechnologies.com
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Safety Data Sheets (SDS)
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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 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 www.lifetechnologies.com/termsandconditions. If you have any questions, please contact Life Technologies at www.lifetechnologies.com/support.
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Purchaser Notification
Limited Use Label License: 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 [email protected] or Out Licensing, Life Technologies, 5791 Van Allen Way, Carlsbad, California 92008.
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References 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). 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. Graham, F. L., Smiley, J., Russell, W. C., and Nairn, R. (1977). Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5. J. Gen. Virol. 36, 59-74. Kao, F. T., and Puck, T. T. (1968). Genetics of Somatic Mammalian Cells, VII. Induction and Isolation of Nutritional Mutants in Chinese Hamster Cells. Proc. Natl. Acad. Sci. USA 60, 1275-1281. Neumann, J. R., Morency, C. A., and Russian, K. O. (1987). A Novel Rapid Assay for Chloramphenicol Acetyltransferase Gene Expression. BioTechniques 5, 444-447. 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, 1551-1557. ©2012 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.
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Notes
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