Introduction

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:

  • Single 3´ thymidine (T) overhangs for TA Cloning®
  • Topoisomerase covalently bound to the vector (this is referred to as “activated” vector)


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.

TOP

Materials

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.

ItemConcentrationAmount
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 MgCl2
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 MgCl2
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.

ItemCompositionAmount
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 MgCl2
10 mM MgSO4
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.

PrimerSequenceAmount
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

TOP

Ordering Information

Experimental Outline

The flow chart below outlines the experimental steps necessary to clone and express your PCR product.




TOP

PCR Primer Design

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:

  • Clone in frame with the V5 epitope and polyhistidine tag (C-terminal peptide) in order to detect and/or purify your PCR product.


OR

  • Include the native stop codon to express the native protein.


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 Web site (www.invitrogen.com) or requested from Technical Service.


TOP

Producing PCR Products

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.

  • Taq polymerase
  • Thermocycler
  • DNA template and primers for PCR product
 
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

  1. Set up the following 50 µl PCR reaction. Use less DNA if you are using plasmid DNA as a template and more DNA if you are using genomic DNA as a template. Use the cycling parameters suitable for your primers and template. Be sure to include a 7 to 30 minute extension at 72°C after the last cycle to ensure that all PCR products are full length and 3´adenylated. 

  2. DNA Template 10-100 ng
    10X PCR Buffer5 µl
    50 mM dNTPs0.5 µl
    Primers 100-200 ng each
    Sterile wateradd to a final volume of 49 µl
    Taq Polymerase (1 unit/µl)1 µl
    Total Volume 50 µl

     
  3. Check the PCR product by agarose gel electrophoresis. You should see a single, discrete band. If you do not see a single band, please refer to the Note below.

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). The PCR Optimizer™ Kit (Catalog no. K1220-01) from Invitrogen can help you optimize your PCR.
TOP

TOPO® Cloning Reaction and Transformation

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.
 
Recent experiments at Invitrogen 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.

TOP

 
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.

  • 42°C water bath (or electroporator with cuvettes, optional)
  • LB plates containing 50-100 µg/ml ampicillin (two for each transformation)
  • Reagents and equipment for agarose gel electrophoresis
  • 37°C shaking and non-shaking incubator


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.
 

  • Equilibrate a water bath to 42°C (for chemical transformation) or set up your electroporator if you are using electrocompetent E. coli.
  • For electroporation, dilute a small portion of the Salt Solution 4-fold to prepare Dilute Salt Solution (e.g. add 5 µl of the Salt Solution to 15 µl sterile water)
  • Warm the vial of SOC medium from Box 2 to room temperature.
  • Warm selective plates at 37°C for 30 minutes.
  • Thaw on ice 1 vial of One Shot® cells for each transformation.

 
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.

TOP

 

Reagent*Chemically Competent E. coliElectrocompetent 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

TOP

 

  1. Mix reaction gently and incubate for 5 minutes at room temperature (22-23°C). Note: For most applications, 5 minutes will yield plenty of colonies for analysis. Depending on your needs, the length of the TOPO® Cloning reaction can be varied from 30 seconds to 30 minutes. For routine subcloning of PCR products, 30 seconds may be sufficient. For large PCR products (> 1 kb) or if you are TOPO® Cloning a pool of PCR products, increasing the reaction time will yield more colonies.

  2. Place the reaction on ice and proceed to One Shot® Chemical Transformation or Transformation by Electroporation. Note: You may store the TOPO® Cloning reaction at -20 °C overnight.

 
One Shot® TOP10 Chemical Transformation

  1. Add 2 µl of the TOPO® Cloning reaction from Step 2 above into a vial of One Shot® TOP10 Chemically Competent E. coli and mix gently. Do not mix by pipetting up and down.

  2. Incubate on ice for 5 to 30 minutes. Note: Longer incubations on ice seem to have a minimal effect on transformation efficiency. The length of the incubation is at the user’s discretion.

  3. Heat-shock the cells for 30 seconds at 42°C without shaking.

  4. Immediately transfer the tubes to ice.

  5. Add 250 µl of room temperature SOC medium.

  6. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37°C for 1 hour.

  7. Spread 25-200 µl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.

  8. An efficient TOPO® Cloning reaction will produce hundreds of colonies. Pick ~10 colonies for analysis (see Analysis of Positive Clones).

 
Transformation by Electroporation

  1.  Add 2 µl of the TOPO® Cloning reaction into a 0.1 cm cuvette containing 50 µl of electrocompetent E. coli and mix gently. Do not mix by pipetting up and down. Avoid formation of bubbles.

  2.  Electroporate your samples using your own protocol and your electroporator. Note:  If you have problems with arcing, see below.

  3. Immediately add 250 µl of room temperature SOC medium.

  4. Transfer the solution to a 15 ml snap-cap tube (i.e. Falcon) and shake for at least 1 hour at 37°C to allow expression of the antibiotic resistance gene.

  5. Spread 10-50 µl from each transformation on a prewarmed selective plate and incubate overnight at 37°C. To ensure even spreading of small volumes, add 20 µl of SOC. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies.

  6. An efficient TOPO® Cloning reaction will produce hundreds of colonies. Pick ~10 colonies for analysis (see Analysis of Positive Clones).

 
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:

  • Reduce the voltage normally used to charge your electroporator by 10%
  • Reduce the pulse length by reducing the load resistance to 100 ohms
  • Ethanol-precipitate the TOPO® Cloning reaction and resuspend in water prior to electroporation

 
Analysis of Positive Clones

  1. Pick 10 colonies and culture them overnight in LB medium containing 50 µg/ml ampicillin (3-5 ml).

  2. Isolate plasmid DNA using your method of choice. If you need ultra-pure plasmid DNA for automated or manual sequencing, we recommend the S.N.A.P.™ MiniPrep Kit (Catalog no. K1900-01).

  3. Please note that PCR products will clone bidirectionally. Analyze the plasmids for insertion and orientation by restriction analysis or by sequencing. The T7 and BGH Reverse sequencing primers are included to help you sequence your insert.


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 Web site (www.invitrogen.com) 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

TOP

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.

 

  1. Prepare a PCR cocktail consisting of PCR buffer, dNTPs, primers, and Taq polymerase. Use a 20 µl reaction volume. Multiply by the number of colonies to be analyzed (e.g. 10).

  2. Pick 10 colonies and resuspend them individually in 20 µl of the PCR cocktail. (Don't forget to make a patch plate to preserve the colonies for further analysis.)

  3. Incubate the reaction for 10 minutes at 94°C to lyse the cells and inactivate nucleases.

  4. Amplify for 20 to 30 cycles using parameters previously determined (see text, above).

  5. For the final extension, incubate at 72°C for 10 minutes. Store at +4°C.

  6. Visualize by agarose gel electrophoresis.

 
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.

  1. Streak the original colony on LB plates containing 50-100 µg/ml ampicillin.

  2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50-100 µg/ml ampicillin. Grow until culture reaches stationary phase.

  3. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.

  4. Store at -80°C.
TOP

Optimizing the TOPO®Cloning Reaction

Introduction

The information below will help you optimize the TOPO® Cloning reaction for your particular needs.
 
Faster Subcloning

The high efficiency of TOPO® Cloning technology allows you to streamline the cloning process. If you routinely clone PCR products and wish to speed up the process, consider the following:

  • Incubate the TOPO® Cloning reaction for only 30 seconds instead of 5 minutes.
  • You may not obtain the highest number of colonies, but with the high cloning efficiency of TOPO® Cloning, most of the transformants will contain your insert.
  • After adding 2 µl of the TOPO® Cloning reaction to chemically competent cells, incubate on ice for only 5 minutes.
  • Increasing the incubation time to 30 minutes does not significantly improve transformation efficiency.
 
More Transformants

If you are TOPO® Cloning large PCR products, toxic genes, or cloning a pool of PCR products, you may need more transformants to obtain the clones you want. To increase the number of colonies:

  • Incubate the salt-supplemented TOPO® Cloning reaction for 20 to 30 minutes instead of 5 minutes.
  • Increasing the incubation time of the salt-supplemented TOPO® Cloning reaction allows more molecules to ligate, increasing the transformation efficiency. Addition of salt appears to prevent topoisomerase from rebinding and nicking the DNA after it has ligated the PCR product and dissociated from the DNA. 
 
Cloning Dilute PCR Products

To clone dilute PCR products, you may:

  • Increase the amount of the PCR product
  • Incubate the TOPO® Cloning reaction for 20 to 30 minutes
  • Concentrate the PCR product   
TOP

Transfection

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). Invitrogen offers 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 Web site (www.invitrogen.com) 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. Invitrogen offers the b-Gal Assay Kit (Catalog no. K1455-01) and the b-Gal Staining Kit (Catalog no. K1465-01) for fast and easy detection of b-galactosidase expression. 
TOP

Expression and Purification

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

A number of antibodies are available from Invitrogen 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. 

AntibodyPurposeCatalog 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).

  1. Seed cells in either five T-75 flasks or 2 to 3 T-175 flasks.

  2. Grow the cells in selective medium until they are 80-90% confluent.

  3. Harvest the cells by treating with trypsin-EDTA for 2 to 5 minutes or by scraping the cells in PBS.

  4. Inactivate the trypsin by diluting with fresh medium (if necessary) and transfer the cells to a sterile microcentrifuge tube.

  5. Centrifuge the cells at 1500 rpm for 5 minutes. You may lyse the cells immediately or freeze in liquid nitrogen and store at -80°C until needed. Note: For cell lysis pro­cedures, refer to the ProBond™ Purification System manual if using ProBond™. If you are using a different resin, refer to the manufacturer's instructions.
TOP

Creation of Stable Cell Lines

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 Invitrogen (Catalog no. 11811-031). Use as follows:

  1. Prepare Geneticin® in a buffered solution (e.g. 100 mM HEPES, pH 7.3).

  2. Use 100 to 1000 µg/ml of Geneticin® in complete medium.

  3. Calculate concentration based on the amount of active drug.

  4. Test varying concentrations of Geneticin® on your cell line to determine the concentration that kills your cells (kill curve). Cells differ in their susceptibility to Geneticin®.

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.
TOP

Detecting Recombinant Fusion Proteins

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:

  1. Wash cell monolayers (~5 x 105 to 1 x 106 cells) once with phosphate-buffered saline.

  2. Scrape cells into 1 ml PBS and pellet the cells at 1500 x g for 5 minutes.

  3. Resuspend in 50 µl Cell Lysis Buffer. Other cell lysis buffers are suitable. Vortex.

  4. Incubate cell suspension at 37°C for 10 minutes to lyse the cells. Note: You may prefer to lyse the cells at room temperature or on ice if degradation of your protein is a potential problem.

  5. Centrifuge the cell lysate at 10,000 x g for 10 minutes at +4°C to pellet nuclei and transfer the supernatant to a fresh tube. Assay the lysate for protein concentration. Note: Do not use protein assays utilizing Coomassie Blue or other dyes. NP-40 interferes with the binding of the dye with the protein.

  6. Add SDS-PAGE sample buffer to a final concentration of 1X and boil the sample for 5 minutes.

  7. Load 20 µg of lysate onto an SDS-PAGE gel and electrophorese. Use the appropriate percentage of acrylamide to resolve your fusion protein.
 
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 Invitrogen. Invitrogen 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, refer to our Web site (www.invitrogen.com) or contact Technical Service.
 
Detecting Fusion Proteins

To detect expression of your recombinant fusion protein by western blot analysis, you may use the Anti-V5 antibodies or the Anti-His(C-term) antibodies available from Invitrogen or an antibody to your protein of interest. In addition, the Positope™ Control Protein (Catalog no. R900-50) is available from Invitrogen 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 available from Invitrogen to facilitate detection of antibodies by colorimetric or chemiluminescent methods. For more information, refer to our Web site (www.invitrogen.com) 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). Invitrogen offers the β-Gal Antiserum, the β-Gal Assay Kit, and the β-Gal Staining Kit for fast and easy detection of β-galactosidase expression. 
TOP

Control Reactions

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:

  • 40 mg/ml X-gal in dimethylformamide
  • LB plates containing 50-100 µg/ml ampicillin and X-gal (two per transformation)
  • To add X-gal to previously made agar plates, warm the plate to 37°C. Pipette 40 µl of the 40 mg/ml stock solution onto the plate, spread evenly, and let dry 15 minutes. Protect plates from light.

 
Producing the Control PCR Product

  1. To produce the 500 bp control PCR product containing the lac promoter and LacZa, set up the following 50 µl PCR:

  2. Control DNA Template (50 ng)                            1 µl
    10X PCR Buffer                                                   5 µl
    50 mM dNTPs                                                    0.5 µl
    Control PCR Primers (0.1 µg/µl each)                  2 µl
    Sterile Water                                                   40.5 µl       
    Taq Polymerase (1 unit/µl)                                  1  µl
    Total Volume                                                      50 µl


  3. Overlay with 70 µl (1 drop) of mineral oil.

  4. Amplify using the following cycling parameters:

    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

     

  5. Remove 10 µl from the reaction and analyze by agarose gel electrophoresis. A discrete 500 bp band should be visible. Proceed to the Control TOPO® Cloning Reactions.



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.

  1. Set up control TOPO® Cloning reactions:

  2. 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

      
  3. Transform 2 µl of each reaction into separate vials of One Shot® TOP10 cells.

  4. Spread 10-50 µl of each transformation mix onto LB plates containing 50-100 µg/ml ampicillin and X-Gal. Be sure to plate two different volumes to ensure that at least one plate has well-spaced colonies. For plating small volumes, add 20 µl of SOC to allow even spreading.

  5. Incubate overnight at 37°C.



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

TOP

Addition of 3´ A-Overhangs Post-Amplification

Introduction

Direct cloning of DNA amplified by Vent® or Pfu polymerases into TOPO TA Cloning® vectors is often difficult because of very low cloning efficiencies. These low efficiencies are caused by the lack of the terminal transferase activity associated with proofreading polymerases which adds the 3´ A-overhangs necessary for TA Cloning®. A simple method is provided below to clone these blunt-ended fragments.
 
Before Starting

You will need the following items:

  • Taq polymerase
  • A heat block equilibrated to 72°C
  • Phenol-chloroform (optional)
  • 3 M sodium acetate (optional)
  • 100% ethanol (optional)
  • 80% ethanol (optional)
  • TE buffer (optional)
 
Procedure

This is just one method for adding 3´ adenines. Other protocols may be suitable.

  1. After amplification with Vent® or Pfu  or Pfx polymerase, place vials on ice and add 0.7-1 unit of Taq polymerase per tube. Mix well. It is not necessary to change the buffer.

  2. Incubate at 72°C for 8-10 minutes (do not cycle).

  3. Place the vials on ice. The DNA amplification product is now ready for ligation into pcDNA3.1/V5-His-TOPO©.

Note:    If you plan to store your sample(s) overnight before proceeding with TOPO® Cloning, you may want to extract your sample(s) with phenol-chloroform to remove the polymerases. After phenol-chloroform extraction, precipitate the DNA with ethanol and resuspend the DNA in TE buffer to the starting volume of the amplification reaction.
 
You may also gel-purify your PCR product after amplification with Vent® or Pfu or Pfx.  After purification, add Taq polymerase buffer, dATP, and 0.5 unit of Taq polymerase and incubate 10-15 minutes at 72°C. Use 4 µl in the TOPO® Cloning reaction.
 
Vent® is a registered trademark of New England Biolabs
TOP

References

  1. Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., and Russell, D. W. (1989). Cloning, Structure, and Expression of the Mitochondrial Cytochrome P-450 Sterol 26-Hydroxylase, a Bile Acid Biosynthetic Enzyme. J. Biol. Chem. 264, 8222-8229.

  2. 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).

  3. Boshart, M., Weber, F., Jahn, G., Dorsch-Häsler, K., Fleckenstein, B., and Schaffner, W. (1985). A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus. Cell 41, 521-530.

  4. Brownstein, M. J., Carpten, J. D., and Smith, J. R. (1996). Modulation of Non-Templated Nucleotide Addition by Taq DNA Polymerase: Primer Modifications that Facilitate Genotyping. BioTechniques 20, 1004-1010.

  5. Chen, C., and Okayama, H. (1987). High-Efficiency Transformation of Mammalian Cells by Plasmid DNA. Molec. Cell. Biol. 7, 2745-2752.

  6. Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian Cells with DNA. Nucleic Acids Res. 15, 1311-1326 

  7. Felgner, P. L., Holm, M., and Chan, H. (1989). Cationic Liposome Mediated Transfection. Proc. West. Pharmacol. Soc. 32, 115-121.

  8. Felgner, P. L., and Ringold, G. M. (1989). Cationic Liposome-Mediated Transfection. Nature 337, 387-388.

  9. 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.

  10. Innis, M. A., Gelfand, D. H., Sninsky, J. J., and White, T. S. (1990) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA.

  11. Lindner, P., Bauer, K., Krebber, A., Nieba, L., Kremmer, E., Krebber, C., Honegger, A., Klinger, B., Mocikat, R., and Pluckthun, A. (1997). Specific Detection of His-tagged Proteins With Recombinant Anti-His Tag scFv-Phosphatase or scFv-Phage Fusions. BioTechniques 22, 140-149.

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

  13. Nelson, J. A., Reynolds-Kohler, C., and Smith, B. A. (1987). Negative and Positive Regulation by a Short Segment in the 5´-Flanking Region of the Human Cytomegalovirus Major Immediate-Early Gene. Molec. Cell. Biol. 7, 4125-4129.

  14. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Second Edition (Plainview, New York: Cold Spring Harbor Laboratory Press). 

  15. Shigekawa, K., and Dower, W. J. (1988). Electroporation of Eukaryotes and Prokaryotes: A General Approach to the Introduction of Macromolecules into Cells. BioTechniques 6, 742-751.

  16. Shuman, S. (1994). Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase. J. Biol. Chem. 269, 32678-32684.

  17. Shuman, S. (1991). Recombination Mediated by Vaccinia Virus DNA Topoisomerase I in Escherichia coli is Sequence Specific. Proc. Natl. Acad. Sci. USA 88, 10104-10108.

  18. 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.

  19. Southern, P. J., and Berg, P. (1982). Transformation of Mammalian Cells to Antibiotic Resistance with a Bacterial Gene Under Control of the SV40 Early Region Promoter. J. Molec. Appl. Gen. 1, 327-339.

  20. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.-C., and Axel, R. (1977). Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells. Cell 11, 223-232.
TOP
LT051