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The pcDNA3.1/V5-His© TOPO® TA Expression Kit provides a highly efficient, 5 minute, one-step cloning strategy ("TOPO® Cloning") for the direct insertion of Taq polymerase-amplified PCR products into a plasmid vector. No ligase, post-PCR procedures, or PCR primers containing specific sequences are required. Once cloned, analyzed, and transfected, the PCR product will express directly in mammalian cell lines.
How It Works
The plasmid vector (pcDNA3.1/V5-His-TOPO®) is supplied linearized with:
Taq polymerase has a nontemplate-dependent terminal transferase activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in this kit has single, overhanging 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
Topoisomerase I from Vaccinia virus binds to duplex DNA at specific sites and cleaves the phosphodiester backbone after 5'-CCCTT in one strand (Shuman, 1991). The energy from the broken phosphodiester backbone is conserved by formation of a covalent bond between the 3' phosphate of the cleaved strand and a tyrosyl residue (Tyr-274) of topoisomerase I. The phospho-tyrosyl bond between the DNA and enzyme can subsequently be attacked by the 5' hydroxyl of the original cleaved strand, reversing the reaction and releasing topoisomerase (Shuman, 1994). TOPO® Cloning exploits this reaction to efficiently clone PCR products (see below).
Once the PCR product is cloned into pcDNA3.1/V5-His-TOPO® and transformants analyzed for the correct orientation of the PCR product, the plasmid is transfected into mammalian cells for expression. The PCR product may be expressed as a fusion to the V5 epitope and polyhistidine tag for detection and purification; or, by designing the 3´ PCR primer with a stop codon, the PCR product may be expressed as a native protein.
Shipping and Storage
The pcDNA3.1/V5-His© TOPO® TA Expression Kit is shipped on dry ice. Each kit contains a box with pcDNA3.1/V5-His TOPO TA Cloning® reagents (Box 1) and a box with One Shot® TOP10 chemically competent cells (Box 2). Store Box 1 at -20°C and Box 2 at -80°C.
Types of Kits
Ordering information for the pcDNA3.1/V5-His© TOPO® TA Expression Kits is provided below.
TOPO TA Cloning® Reagents
pcDNA3.1/V5-His TOPO TA Cloning® reagents (Box 1) are listed below. Please note that the user must supply Taq polymerase.
Store Box 1 at -20°C.
Item | Concentration | Amount |
pcDNA3.1/V5-His-TOPO
® |
10 ng/µl plasmid DNA in:
50% glycerol
50 mM Tris-HCl, pH 7.4 (at 25°C)
1 mM EDTA
2 mM DTT
0.1% Triton X-100
100 µg/ml BSA
30 µM phenol red
|
20 µl
|
10X PCR Buffer
|
100 mM Tris-HCl, pH 8.3 (at 42°C)
500 mM KCl
25 mM MgCl
2
0.01% gelatin
|
100 µl
|
50 mM dNTPs
|
12.5 mM dATP; 12.5 mM dCTP; 12.5 mM dGTP; 12.5 mM dTTP
neutralized at pH 8.0 in water
|
10 µl
|
Salt Solution
|
1.2 M NaCl; 0.06 M MgCl
2 |
50 µl
|
T7 Sequencing Primer
|
0.1 µg/µl in TE Buffer
|
20 µl
|
BGH Reverse Sequencing Primer
|
0.1 µg/µl in TE Buffer
|
20 µl
|
Control PCR Template
|
0.05 µg/µl in TE Buffer
|
10 µl
|
Control PCR Primers
|
0.1 µg/µl each in TE Buffer
|
10 µl
|
Expression Control Plasmid
|
0.5 µg/µl in TE Buffer
|
10 µl
|
Sterile Water
|
--
|
1 ml
|
One Shot® Reagents
The table below describes the items included in the One Shot® chemically competent cell kit. Store at -80°C.
Item | Composition | Amount | |
SOC Medium
(may be stored at +4°C or room temperature)
|
2% Tryptone
0.5% Yeast Extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl
2
10 mM MgSO
4
20 mM glucose
|
6 ml
| |
TOP10 cells
|
--
|
21 x 50 µl
| |
pUC19 Control DNA
|
10 pg/µl in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8
|
50 µl
|
Sequencing Primers
The table below provides the sequence and pmoles of the T7 sequencing primer and the BGH Reverse sequencing primer.
Primer | Sequence | Amount | |
T7
|
5´-TAATACGACTCACTATAGGG-3´
|
328 pmoles
| |
BGH Reverse
|
5´-TAGAAGGCACAGTCGAGG-3´
|
358 pmoles
|
Genotype of TOP10 Cells
TOP10: Use this strain for general cloning. Please note that this strain cannot be used for single-strand rescue of DNA.
F- mcrA D(mrr-hsdRMS-mcrBC) F80lacZDM15 DlacC74 recA1 araD139 D(ara-leu)7697 galU galK rpsL (StrR) endA1 nupG
The flow chart below outlines the experimental steps necessary to clone and express your PCR product.
Designing Your PCR Primers
Design of the PCR primers to clone your DNA sequences of interest is critical for expression. This is a C-terminal fusion vector that does not contain an ATG initiation codon. If there is no initiating ATG codon or optimal sequences for translation initiation (Kozak sequences) in the DNA to be amplified, then these features need to be incorporated into your forward primer.
Example: Kozak consensus sequence is (G/A)NNATGG
Depending on the nature of your PCR product you have two options to consider:
OR
Note: Cloning efficiencies may vary depending on the 5´ primer nucleotide sequence.
Use the diagram below to design your PCR primers.
Do not add 5´ phosphates to your primers for PCR. The PCR product synthesized will not ligate into pcDNA3.1/V5-His-TOPO®.
TOPO TA Cloning® Site
Restriction sites are labeled to indicate the actual cleavage site. The vector is supplied linearized between base pair 953 and 954. This is the TOPO® Cloning site. Please note that the full sequence of pcDNA3.1/V5-His-TOPO® may be downloaded from our website or requested from Technical Service.
Introduction
Once you have decided on a PCR strategy and have synthesized the primers you are ready to produce your PCR product.
Materials Supplied by the User
You will need the following reagents and equipment.
Polymerase Mixtures
If you wish to use a mixture containing Taq polymerase and a proofreading polymerase, Taq must be used in excess of a 10:1 ratio to ensure the presence of 3´ A-overhangs on the PCR product (i.e. Expand™ or eLONGase™).
If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3' A-overhangs.
Producing PCR Products
DNA Template | 10-100 ng |
10X PCR Buffer | 5 µl |
50 mM dNTPs | 0.5 µl |
Primers | 100-200 ng each |
Sterile water | add to a final volume of 49 µl |
Taq Polymerase (1 unit/µl) | 1 µl |
Total Volume | 50 µl |
If you do not obtain a single, discrete band from your PCR, you may gel-purify your fragment before using the pcDNA3.1/V5-His© TOPO® TA Expression Kit (see page 16). Take special care to avoid sources of nuclease contamination and long exposure to UV light. Alternatively, you may optimize your PCR to eliminate multiple bands and smearing (Innis et al., 1990). Our PCR Optimizer™ Kit (Catalog no. K1220-01) can help you optimize your PCR.
Introduction
TOPO® Cloning technology allows you to produce your PCR products, ligate them into pcDNA3.1/V5-His-TOPO®, and transform the recombinant vector into TOP10 E. coli in one day. It is important to have everything you need set up and ready to use to ensure that you obtain the best possible results. If this is the first time you have TOPO® Cloned, perform the control reactions in parallel with your samples.
Our recent experiments demonstrate that inclusion of salt (200 mM NaCl, 10 mM MgCl2) in the TOPO® Cloning reaction increases the number of transformants 2- to 3-fold. We have also observed that in the presence of salt, incubation times of greater than 5 minutes can also increase the number of transformants. This is in contrast to earlier experiments without salt where the number of transformants decreases as the incubation time increases beyond 5 minutes.
Inclusion of salt allows for longer incubation times because it prevents topoisomerase I from rebinding and potentially nicking the DNA after ligating the PCR product and dissociating from the DNA. The result is more intact molecules leading to higher transformation efficiencies.
Because of the above results, we recommend adding salt to the TOPO® Cloning reaction. A stock salt solution is provided in the kit for this purpose. Please note that the amount of salt added to the TOPO® Cloning reaction varies depending on whether you plan to transform chemically competent cells (provided) or electrocompetent cells (see below). For this reason two different TOPO® Cloning reactions are provided to help you obtain the best possible results. Please read the following information carefully.
Chemically Competent E. coli
For TOPO® Cloning and transformation into chemically competent E. coli, adding sodium chloride and magnesium chloride to a final concentration of 200 mM NaCl, 10 mM MgCl2 in the TOPO® Cloning reaction increases the number of colonies over time. A Salt Solution (1.2 M NaCl; 0.06 M MgCl2) is provided to adjust the TOPO® Cloning reaction to the recommended concentration of NaCl and MgCl2.
Electrocompetent E. coli
For TOPO® Cloning and transformation of electrocompetent E. coli, salt must also be included in the TOPO® Cloning reaction, but the amount of salt must be reduced to 50 mM NaCl, 2.5 mM MgCl2 to prevent arcing. The Salt Solution is diluted 4-fold to prepare a 300 mM NaCl, 15 mM MgCl2 solution for convenient addition to the TOPO® Cloning reaction.
Materials Supplied by the User
In addition to general microbiological supplies (i.e. plates, spreaders), you will need the following reagents and equipment.
There is no blue-white screening for the presence of inserts. Individual recombinant plasmids need to be analyzed by restriction analysis or sequencing for the presence and orientation of insert. Sequencing primers included in the kit can be used to sequence across an insert in the multiple cloning site to confirm orientation and reading frame.
Preparation for Transformation
For each transformation, you will need one vial of competent cells and two selective plates.
Setting Up the TOPO® Cloning Reaction
The table below describes how to set up your TOPO® Cloning reaction (6 µl) for eventual transformation into either chemically competent One Shot® TOP10 E. coli (provided) or electrocompetent E. coli.
Note: The red or yellow color of the TOPO® vector solution is normal and is used to visualize the solution.
Reagent* | Chemically Competent E. coli | Electrocompetent E. coli |
Fresh PCR product
|
0.5 to 4 µl
|
0.5 to 4 µl
|
Salt Solution
|
1 µl
|
--
|
Dilute Salt Solution
|
--
|
1 µl
|
Sterile Water
|
add to a final volume of 5 µl
|
add to a final volume of 5 µl
|
TOPO
® vector
|
1 µl
|
1 µl
|
*Store all reagents at -20 °C when finished. Salt solutions and water can be stored at room temperature or +4 °C.
Performing the TOPO® Cloning Reaction
One Shot® TOP10 Chemical Transformation
Transformation by Electroporation
Addition of the Dilute Salt Solution in the TOPO® Cloning Reaction brings the final concentration of NaCl and MgCl2 in the TOPO® Cloning reaction to 50 mM and 2.5 mM, respectively. To prevent arcing of your samples during electroporation, the volume of cells should be between 50 and 80 µl (0.1 cm cuvettes) or 100 to 200 µl (0.2 cm cuvettes).
If you experience arcing during transformation, try one of the following suggestions:
Analysis of Positive Clones
Please refer to the diagram for restriction sites and sequence surrounding the TOPO Cloning® site. For the complete sequence of the vector, please see our website or contact Technical Service.
If you need help with setting up restriction enzyme digests or DNA sequencing, please refer to general molecular biology texts (Ausubel et al., 1994; Sambrook et al., 1989).
Alternative Method of Analysis
You may wish to use PCR to directly analyze positive transformants. Use a combination of either the T7 or the BGH Reverse sequencing primer with a primer that binds within your insert as PCR primers. You will have to determine the amplification conditions. If this is the first time you have used this technique, we recommend that you perform restriction analysis in parallel to confirm that PCR gives you the correct result. Artifacts may be obtained because of mispriming or contaminating template.
The following protocol is provided for your convenience. Other protocols are suitable.
If you have problems obtaining transformants or the correct insert, perform the control reactions. These reactions will help you troubleshoot your experiment.
Long-Term Storage
Once you have identified the correct clone, be sure to isolate a single colony and prepare a glycerol stock for long term storage. We recommend that you store a stock of plasmid DNA at -20°C.
Introduction
Once you have the desired construct, you are ready to transfect the plasmid into the mammalian cells of choice. Please note the following guidelines for transfection. Included in the kit is an expression control vector (pcDNA3.1/V5-His-TOPO/lacZ©) which you can use to check both transfection efficiencies and expression in your particular cell line.
Plasmid Preparation
Plasmid DNA for transfection into eukaryotic cells must be very clean and free from phenol and sodium chloride. Contaminants will kill the cells and salt will interfere with lipids decreasing transfection efficiency. We recommend isolating plasmid DNA (up to 200 µg) using the S.N.A.P.™ MidiPrep Kit (Catalog no. K1910-01) or CsCl gradient centrifugation.
Methods of Transfection
For established cell lines (e.g. HeLa), please 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). We offer the Calcium Phosphate Transfection Kit (Catalog no. K2780-01) and a large selection of reagents for transfection. For more information on the reagents available, please visit our website or call Technical Service.
Positive Control
pcDNA3.1/V5-His-TOPO/lacZ© is provided as a positive control vector for mammalian transfection and expression. It may be used to optimize transfection conditions for your cell line. The gene encoding b-galactosidase is expressed in mammalian cells under the CMV promoter. A successful transfection will result in ß-galactosidase expression that can be easily assayed (see below).
Assay for ß-galactosidase Activity
You may assay for b-galactosidase expression by activity assay using cell-free lysates (Miller, 1972) or by staining the cells for activity. We offer the b-Gal Assay Kit (Catalog no. K1455-01) and the b-Gal Staining Kit (Catalog no. K1465-01) for fast and easy detection of b-galactosidase expression.
Introduction
Expression of your PCR product can be performed in either transiently transfected cells or stable cell lines. You may use a functional assay to detect the protein encoded by your PCR product or a Western blot analysis if you have an antibody to the protein. If you have elected to express your PCR product as a fusion to the V5 epitope and the polyhistidine tag, you may use antibodies to the V5 epitope or the polyhistidine C-terminus to detect the fusion protein. If you wish, the fusion protein may be purified using metal ion chromatography (see below).
Detection of Fusion Proteins
To detect the fusion protein by Western blot, you will need to prepare a cell lysate from transfected cells. We recommend that you perform a time course to optimize expression of the fusion protein (e.g. 24, 48, 72 hours, etc. after transfection).
The C-terminal peptide containing the V5 epitope and the polyhistidine region will add approximately ~5 kDa to your protein.
Antibodies for Detection
We offer a number of antibodies to detect expression of your fusion protein from pcDNA3.1/V5-His-TOPO©. The table below describes the antibodies available and ordering information. The amount supplied is sufficient for 25 Westerns.
Antibody | Purpose | Catalog no. | |
Anti-V5
|
Detects 14 amino acid epitope derived from the P and V proteins of the paramyxovirus, SV5 (Southern
et al., 1991)
|
R960-25
| |
Anti-V5-HRP
|
See above. Provided as an HRP conjugate for time-saving detection.
|
R961-25
| |
Anti-His(C-term)
|
Detects the C-terminal polyhistidine tag (requires the free carboxyl group for detection) (Lindner
et al., 1997)
|
R930-25
| |
Anti-His(C-term)-HRP
|
See above. Provided as an HRP conjugate for time-saving detection.
|
R931-25
|
Preparing Cells for Purification
Use the procedure below to prepare cells for lysis prior to purification of your protein on ProBond™. You will need 5 x 106 to 1 x 107 cells for purification of your protein on a 2 ml ProBond™ column (see ProBond™ Purification System manual).
Introduction
If you wish to create stable cell lines, select for foci using Geneticin® Selective Antibiotic. General information and guidelines are provided below for your convenience.
Geneticin® Selective Antibiotic
Geneticin® Selective Antibiotic blocks protein synthesis in mammalian cells by interfering with ribosomal function. It is an aminoglycoside, similar in structure to neomycin, gentamycin, and kanamycin. Expression in mammalian cells of the bacterial aminoglycoside phosphotransferase gene (APH), derived from Tn5, results in detoxification of Geneticin® (Southern and Berg, 1982).
Geneticin® Selection Guidelines
Geneticin® Selective Antibiotic is available from Thermo Fisher Scientific (Catalog no. 11811-031). Use as follows:
Cells will divide once or twice in the presence of lethal doses of Geneticin®, so the effects of the drug take several days to become apparent. Complete selection can take from 2 to 4 weeks of growth in selective medium.
Introduction
You may express you gene of interest in either transiently transfected cells or 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:
Polyacrylamide Gel Electrophoresis
To facilitate separation and visualization of your recombinant fusion protein by polyacrylamide gel electrophoresis, we offer a wide range of pre-cast NuPAGE® and Novex® Tris-Glycine polyacrylamide gels and electrophoresis apparatus. We also carry 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, refer to our website or contact Technical Service.
Detecting Fusion Proteins
To detect expression of your recombinant fusion protein by western blot analysis, you may use our Anti-V5 antibodies, Anti-His(C-term) antibodies or an antibody to your protein of interest. In addition, the Positope™ Control Protein (Catalog no. R900-50) is available from us for use as a positive control for detection of fusion proteins containing a V5 epitope or a polyhistidine (6xHis) tag. The ready-to-use WesternBreeze® Chromogenic Kits and WesternBreeze® Chemiluminescent Kits are also available to facilitate detection of antibodies by colorimetric or chemiluminescent methods. For more information, refer to our website or contact Technical Service.
The C-terminal peptide containing the V5 epitope and the polyhistidine region will add approximately 3.6 kDa to your protein.
Assay for β-galactosidase Activity
If you use the expression control plasmid, you may assay for β-galactosidase expression by Western blot analysis or activity assay using cell-free lysates (Miller, 1972). We offer the β-Gal Antiserum, the β-Gal Assay Kit, and the β-Gal Staining Kit for fast and easy detection of β-galactosidase expression.
pcDNA3.1/V5-His© TOPO® TA Cloning Control Reactions
Introduction
We recommend performing the following control TOPO® Cloning reactions the first time you use the kit to help you evaluate your results. Performing the control reactions using the reagents included in the kit involves producing a control PCR product containing the lac promoter and the LacZa protein. Successful TOPO® Cloning of the control PCR product will yield blue colonies on LB agar plates containing ampicillin and X-gal.
Before Starting
Be sure to prepare the following reagents before performing the control reaction:
Producing the Control PCR Product
Step | Time | Temperature | Cycles | ||
Initial Denaturation
|
2 minutes
|
94°C
|
1X
| ||
Denaturation
|
1 minute
|
94°C
| |||
Annealing
|
1 minute
|
60°C
|
25X
| ||
Extension
|
1 minute
|
72°C
| |||
Final Extension
|
7 minutes
|
72°C
|
1X
|
Control TOPO® Cloning Reactions
Using the control PCR product produced and the pcDNA3.1/V5-His-TOPO© vector set up two 6 µl TOPO® Cloning reactions as described below.
Reagent | "Vector Only" | "Vector + PCR Insert" | ||
Sterile Water
|
4 µl
|
3 µl
| ||
Salt Solution or Dilute Salt Solution
|
1 µl
|
1 µl
| ||
Control PCR Product
|
--
|
1 µl
| ||
pcDNA3.1/V5-His-TOPO
© vector
|
1 µl
|
1 µl
|
Analysis of Results
Hundreds of colonies from the vector + PCR insert reaction should be produced. Greater than 90% of these will be blue and contain the 500 bp insert.
Transformation Control
pUC19 plasmid is included to check the transformation efficiency of the One Shot® TOP10 competent cells. Transform one vial of One Shot® TOP10 cells with 10 pg of pUC19 using the protocol. Plate 10 µl of the transformation mixture plus 20 µl SOC on LB plates containing 100 µg/ml ampicillin. Transformation efficiency should be ~1 x 109 cfu/µg DNA.
Factors Affecting Cloning Efficiency
Please note that lower transformation and/or cloning efficiencies will result from the following variables. Most of these are easily corrected, but if you are cloning large inserts, you may not obtain the expected 90% (or more) cloning efficiency.
Variable | Solution |
pH>9
|
Check the pH of the PCR amplification reaction and adjust with 1 M Tris-HCl, pH 8.
|
Incomplete extension during PCR
|
Be sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
|
Cloning large inserts (>3 kb)
|
Gel-purify as described.
|
Excess (or overly dilute) PCR product
|
Reduce (or concentrate) the amount of PCR product.
|
Cloning blunt-ended fragments
|
Add 3´ A-overhangs by incubating with
Taq polymerase.
|
PCR cloning artifacts ("false positives")
|
TOPO
® Cloning is very efficient for small fragments (< 100 bp) present in certain PCR reactions. Gel-purify your PCR product.
|
PCR product does not contain sufficient 3´ A-overhangs even though you used
Taq polymerase
| Taq polymerase is less efficient at adding a nontemplate 3´ A next to another A.
Taq is most efficient at adding a nontemplate 3´ A next to a C. You may have to redesign your primers so that they contain a 5´ G instead of a 5´ T (Brownstein
et al., 1996).
|
Incubate at room temperature for 5 minutes and place on ice