Introduction

Description of the System

TOPO® XL PCR Cloning provides a highly efficient, 5 minute, one-step cloning strategy ("TOPO® Cloning") for the cloning of long PCR products generated by commercial enzyme mixes specifically formulated to generate long PCR products (e.g. Elongase™, available from Invitrogen). No ligase, or PCR primers containing specific sequences are required. Special gel purification reagents are provided to ensure efficient cloning of long, full-length PCR products.

How It Works

The plasmid vector (pCR®-XL-TOPO®) is supplied linearized with:

  • Single 3´ thymidine (T) overhangs for TA Cloning®
  • Topoisomerase covalently bound to the plasmid (referred to as "activated" vector)

Most commercial enzyme mixtures designed to generate long PCR products contain mainly Taq polymerase. 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). The TOPO® Cloning Reaction can be transformed directly into competent cells (provided) by either electroporation or chemical means (see Recommendation, below).

Selection of Recombinants

pCR®-XL-TOPO® allows direct selection of recombinants via disruption of the lethal E. coli gene, ccdB (Bernard et al., 1994). The vector contains the ccdB gene fused to the C-terminus of the LacZα fragment. Ligation of a long PCR product disrupts expression of the lacZα-ccdB gene fusion permitting growth of only positive recombinants upon transformation. Cells that contain non-recombinant vector are killed upon plating. Therefore, blue/white screening is not required. Lastly, by gel-purifying and visualizing the PCR product on agarose gels containing crystal violet, the damaging effects of ethidium bromide in the presence of UV light are avoided leading to higher cloning efficiencies.

We recommend electroporation as the transformation method of choice. Electroporation increases transformation efficiency of large plasmids (>10 kb) over chemically competent transformation.


Experimental Outline

The chart below outlines the experimental steps necessary to clone your long PCR product.

Amplify your long PCR product



Gel-Purify PCR product using agarose gel electrophoresis with crystal violet



TOPO-Cloning
® Reaction:
Mix together purified PCR product and pCR
® - XL-TOPO®



Incubate 5 minutes at room temp



Transform into competent E. coli cells



Select and analyze colonies
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Ordering Information

Sku Name Size Price Qty
K470010 TOPO® XL PCR Cloning Kit, with One Shot® TOP10 Electrocomp™ E. coli 10 reactions USD 297.00
K470020 TOPO® XL PCR Cloning Kit, with One Shot® TOP10 Electrocomp™ E. coli 20 reactions USD 508.00
K475010 TOPO® XL PCR Cloning Kit, with One Shot® TOP10 Chemically Competent E. coli 10 reactions USD 297.00
K475020 TOPO® XL PCR Cloning Kit, with One Shot® TOP10 Chemically Competent E. coli 20 reactions USD 508.00
K703020 TOPO® XL PCR Cloning Kit, with One Shot® Mach1™ T1 Phage-Resistant Chemically Competent E. coli 20 reactions USD 526.00

Producing PCR Products

Introduction

This kit is specifically designed to clone long PCR products generated by enzyme mixtures such as Elongase™ Enzyme Mix. The first time you use this kit, we recommend performing the control TOPO® Cloning reaction to generate a 7 kb PCR product that can be used as a marker during agarose gel electrophoresis and to help you evaluate your results.

Note:  We have tested this kit specifically with the Elongase™ Enzyme Mix (see ordering information). Other kits for producing long PCR products are suitable. Any enzyme mixtures that you use (commercial or homemade) should contain mainly Taq polymerase (>10:1 unit ratio) to ensure the addition of 3´ A-overhangs.
    
Do not add 5´ phosphates to your primers for PCR. The PCR product synthesized will not ligate into pCR®-XL-TOPO®.

Materials Supplied by the User

  • Enzyme mixtures specially formulated for long PCR (e.g. Elongase™)
  • Thermocycler
  • DNA template and primers for PCR product
  • Apparatus for agarose minigel electrophoresis with 8 lane or 12 lane comb
  • General purpose agarose
  • Ethidium bromide
  • 1X TAE buffer (50 mM Tris-acetate, pH 8, 1 mM EDTA). DO NOT USE TBE. TBE will interfere with DNA isolation using sodium iodide

Producing PCR Products

Set up a 50 μL or 100 μL PCR reaction using the guidelines below:

  • Follow the manufacturer's instructions and recommendations for producing PCR products. Optimize conditions to produce your PCR product because small PCR artifacts will preferentially clone over long PCR products
  • Use the cycling parameters suitable for your primers and template. Make sure to optimize PCR conditions to produce a single, discrete PCR product.
  • Use a 7 to 30 minute final extension to ensure that all PCR products are completely extended.
  • After cycling, place the tube on ice or store at –20ºC for up to 2 weeks. Proceed to Checking the PCR Product, below.

Producing the Control PCR Product

The control product may be produced using buffers from the Elongase™ Amplification System. Use the manual hot start described below in order to maximize production of PCR product. If you use another commercial mixture or a homemade mixture, you may have to optimize conditions.

  1. To produce the 7 kb control PCR product, set up the following PCR in a 0.7 mL PCR tube. Use 5 μL of Elongase™ 5X Buffer A and 5 μL of 5X Buffer B. Final Mg2+ concentration is 1.5 mM.

    PCR Buffer (Elongase™)see above
    XL Control PCR Template (25 ng/μL)1 μL
    50 mM dNTPs0.5 μL
    XL Control PCR Primers (0.2 μg/μL)1 μL
    Sterile Waterto a total volume of 49 μL
    Final Volume49 μL



  2. For a manual hot start, begin the program listed below and pause when the heat block reaches 94°C.

    Step Time
    Temperature
    Cycles
    Initial Denaturation 2 minutes
    94°C 1X
    Denaturation15 seconds
    94°C
    Annealing 1 minute
    56°C 25X
    Extension5 minutes
    68°C
    Final Extension
    7 minutes
    72°C1X


  3. Place the tube in the block for 30 seconds.

  4. Mix in 1 μL of Elongase™ polymerase mix, add 1 drop of mineral oil (if necessary), and continue the program

  5. When program is complete, place the tube on ice.

Checking the PCR Product

Remove 5 to 10 μL from each PCR amplification and analyze by agarose gel electrophoresis. Be sure you have a single, discrete band of the correct size. The control PCR amplification should produce a discrete 7 kb band and >1 μg total of PCR product.
   
It is important to generate sufficient PCR product (>0.2 μg total) to allow for losses during the following purification steps. Pool and concentrate several amplification reactions by ethanol precipitation, if necessary. Proceed to Gel-Purifying PCR Products (below).

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Gel Purifying PCR Products

We recommend gel-purifying long PCR products to increase the cloning efficiency. However, in traditional ethidium bromide agarose gel electrophoresis, exposure of DNA to UV light may damage your DNA and significantly decrease cloning efficiency. To avoid damage to long PCR products by UV light, visualize and purify PCR products by agarose gel electrophoresis using crystal violet which is supplied in the kit (Rand, 1996). The PCR product may also be visualized under normal light as a thin blue band. PCR products can be visualized while the gel is running and excised as soon as they are sufficiently resolved.

Crystal violet is listed as a possible carcinogen, but at the concentrations supplied in the kit, it does not require a Safety Data Sheet (SDS). In addition, crystal violet and sodium iodide will stain skin and fabric. Be sure to wear gloves and a laboratory coat when using these reagents.

Crystal violet is not as sensitive as ethidium bromide. We recommend that you only use this staining method for gel-purification of DNA fragments destined to be cloned. Two hundred nanograms are just visible on an agarose gel containing crystal violet. For isolating long PCR products, we recommend that you load greater than 200 ng per lane.

DO NOT USE
loading buffers containing bromophenol blue or xylene cyanol because the dyes will react with crystal violet and the DNA bands may become distorted. Use the 6X Crystal Violet Loading Buffer supplied in the kit.

Molecular Weight Markers

If you perform the control PCR reaction, you can use the 7 kb control PCR fragment as a molecular weight marker. Alternatively, 4 μg of a lambda Hind III digest mixed with 6X Crystal Violet Loading Buffer will yield visible 4.4 kb, 6.5 kb, 9.4 b, and 23 kb bands for sizing of your PCR product.
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Materials Needed
  • Control PCR product (above)
  • Apparatus for agarose minigel electrophoresis is with 8-lane or 12-lane comb
  • General purpose agarose
  • 1X TAE buffer (50 mM Tris-acetate, pH 8.0, 1 mM EDTA)
  • Clean glass flask
  • Autoclaved water or TE buffer
  • Sterile 15 mL bottle to prepare 1X Final Wash (see next page)
  • New razor blade
  • 42°C to 50°C water bath
  • Microcentrifuge

Before Starting

  • Bring the Sodium Iodide solution, Binding Buffer, 4X Final Wash, and TE Buffer to room temperature. Mix well to re-dissolve salts before using. Store these solutions at room temperature after the first use.
  • To prepare 1X Final Wash, take a sterile 15 mL bottle and transfer all of the 4X Final Wash solution to the bottle. Add 7.5 mL of 100% ethanol to the 4X Final Wash solution to prepare the 1X Final Wash solution. Store at room temperature.

Nuclease Control


It is very important to minimize the presence of nucleases to ensure the highest cloning efficiencies. Follow the guidelines listed below. While some guidelines may not appear as rigorous as others, they are sufficient for purifying long PCR fragments.

  • Wear gloves at all times
  • Use sterile plasticware and glassware
  • Autoclave TAE to use as the running buffer
  • Rinse agarose gel apparatus and comb with autoclaved water or TE buffer
  • Use a new razor to excise gel slice*
  • Use new plastic wrap (e.g. Saran® Wrap) if needed

*The same razor may be used to excise different bands in the same gel if you are careful not to bring over pieces from an earlier excision.

Preparing the Gel

Follow the instructions below to prepare a 0.8% agarose gel. The recipe makes one agarose gel with a volume of 50 mL.

  1. Mix 0.4 g of general purpose agarose and 50 mL 1X TAE buffer in a clean glass flask.

  2. Place flask in the microwave and heat until just boiling. Swirl to dissolve agarose and continue to heat in this fashion for 3 minutes to destroy nucleases.

  3. Remove from the microwave and allow to cool for 3 minutes.

  4. Add 20–40 μL of the 2 mg/mL Crystal Violet solution to the agarose and swirl to mix. The agarose should be light to medium purple in color.

  5. Rinse the gel box and comb with autoclaved water or TE buffer. Note: Use a comb that will hold all of the PCR amplification (48 μL) in one well, if possible.

  6. Pour the gel and set the comb in the gel.

  7. When the gel has solidified, cover the gel with 1X TAE buffer. You do not have to add crystal violet to the running buffer. Proceed to the next section to prepare your sample.

Loading and Running the Gel

  1. Add 8 μL of 6X Crystal Violet Loading Buffer to 40 μL of the PCR amplification and load onto the gel. Prepare the control PCR product in the same manner.

  2. Run the gel at 80 volts until the crystal violet in the gel has run about a quarter of the way UP the gel (crystal violet appears to migrate towards the negative pole). You should also see the thin blue PCR product move down into the gel. If no PCR product is visible, insufficient DNA was loaded (<200 ng).

  3. Compare your PCR product to the 7 kb control PCR fragment. If your PCR product is sufficiently resolved so that you can easily excise the fragment, turn off the power supply. Proceed directly to Excising the PCR Product, below.

Excising the PCR Product

Remember to excise the control fragment as well as your band.

  1. Pour off the buffer (or transfer the gel to new Saran® Wrap). Note: Placing the gel on a fluorescent light box may help visualize the fragments.

  2. Using a new razor blade, carefully excise the desired band from the gel. Note: Razor blade may be rinsed with autoclaved water or TE prior to cutting the next band.

  3. Cut up the excised piece of agarose into small chunks and transfer to a sterile 1.5 mL microcentrifuge tube. Note: Cutting up the agarose piece reduces the time and temperature required to melt the agarose.

  4. Estimate the volume of the agarose pieces (generally this is around 100 μL). Alternatively, you can weigh the gel slice and assume that 1 mg ~ 1 μL.

  5. Add 2.5 times its volume of 6.6 M sodium iodide (i.e. 250 μL) and mix by shaking vigorously by hand or vortexing.

  6. Incubate at 42°C to 50°C until the agarose is completely melted (~2 minutes). Mix the solution periodically by shaking vigorously.

  7. Place the tube at room temperature and add 1.5 volumes of Binding Buffer (i.e., 525 μL) and mix well. Proceed directly to Isolating the PCR Product, below.

Isolating the PCR Product

  1. Assemble a S.N.A.P™ purification column (A) and collection vial (B) and load all of the mixture from Step 7, above, onto the column (875 μL).

  2. Centrifuge at 2,000 to 3,000 × g in a microcentrifuge for 30 seconds at room temperature.

  3. Pour the liquid in the collection vial back onto the column and repeat Step 2.

  4. Repeat Step 3 one more time to bind all the DNA to the column (i.e. load solution onto the column for a total of 3 times).

  5. After the last centrifugation, discard the liquid in the collection tube.

  6. Add 400 μL of 1X Final Wash to the S.N.A.P™ column and centrifuge as in Step 2.

  7. Repeat Step 6 and discard the liquid in the collection tube after the final centrifugation (800 μL).

  8. Centrifuge the column again at maximum speed (>10,000 × g) for at least 1 minute to dry the column resin. Discard the collection vial.

  9. Transfer the column to a new, sterile 1.5 mL microcentrifuge tube.

  10. Add 40 μL of TE buffer directly to the column material and incubate for 1 minute at room temperature to let the buffer absorb into the column.

  11. Centrifuge the column at maximum speed (>10,000 × g) for 1 minute to elute the DNA into the microcentrifuge tube.

  12. Place the tube on ice and discard the column.

  13. Assay 10 μL by ethidium bromide agarose gel electrophoresis to estimate the DNA concentration. Concentration should be between 2 and 40 ng/μL. In most cases, there is no need to concentrate the DNA further.

Store the DNA at –20°C for up to one week or proceed directly to Setting Up the= TOPO® Cloning Reaction, below. For the highest efficiencies, proceed directly to TOPO® Cloning.
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Setting Up the TOPO® Cloning Reaction

Introduction

Once you have gel-purified your PCR product, you are ready to TOPO® Clone it into the pCR®-XL-TOPO® vector and transform the recombinant vector into competent E. coli. It is important to have everything you need set up and ready to use to ensure that you obtain the best possible results. We suggest that you read this section and the section entitled Transforming One Shot® Competent Cells (above) before beginning.

TOPO® Cloning Reaction

  1. Set up the following 5 μL TOPO® Cloning reaction in a sterile microcentrifuge tube. Remember to set up a reaction for the control PCR product. You may also wish to set up a "vector-only" reaction as a negative control (use TE buffer instead of insert).

    Gel-purified long PCR product from Step 12, Isolating the PCR Product                      4 μL
    pCR®-XL-TOPO® vector                                                                                            1 μL
    Final Volume                                                                                                           5 μL
  2. Mix gently and incubate for a minimum of 5 minutes to 30 minutes at room temperature (~25°C).  Note: Extending the incubation time up to 30 minutes may increase cloning efficiency for long PCR products.
  3. Following incubation, add 1 μL of the 6X TOPO Cloning Stop Solution and mix for several seconds at room temperature.
    Note:   Addition of the Stop Solution increases the yield of transformants by an average of 2-fold for chemically competent cells and 10-fold for electrocompetent cells.
  4. Briefly centrifuge the tube and place on ice.


Note:
The TOPO® Cloning reaction may be stored on ice or frozen at –20°C for up to 24 hours. You may see a decrease in the transformation efficiency, but the cloning efficiency should remain high. We recommend that you proceed immediately to Transforming Competent Cells below.

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Transforming Competent Cells

Introduction

Once you have performed the TOPO® Cloning reaction, transform your pCR®- XL-TOPO® construct into competent E. coli provided with your kit. Protocols to transform chemically competent and electrocompetent E. coli are provided below.

Control Reaction

The 7 kb control PCR product contains the ampicillin resistance gene. To estimate cloning efficiency, you will need to prepare LB plates containing both 50 μg/mL kanamycin and 50 μg/mL ampicillin. By plating equal amounts of the transformation mix on plates containing either kanamycin (total transformants) or kanamycin and ampicillin (recombinant transformants), you will be able to estimate cloning efficiency.

Materials Needed

In addition to general microbiological supplies (e.g. plates, spreaders), you will need the following reagents and equipment.

  • Electroporator (for transformation of electrocompetent cells)
  • cm cuvettes for electroporation (for transformation of electrocompetent cells)
  • LB plates containing 50 μg/mL kanamycin or Low Salt LB plates containing 25 μg/mL Zeocin™
  • Note: For the control reaction you will need an additional LB plate containing 50 μg/mL kanamycin and 50 μg/mL ampicillin
  • 37°C shaking and non-shaking incubator
  • 42°C water bath (for transformation of chemically competent cells)


Preparing for Transformation

For each transformation, you will need one vial of competent cells and two selective plate.

  • Thaw the vial of S.O.C. medium from Box 2 and bring to room temperature.
  • Warm selective plates at 37°C for 30 minutes.
  • Thaw on ice 1 vial of One Shot® competent cells for each transformation.
  • For electroporation, place 0.1 cm cuvettes on ice.


pUC19 Transformation Control

pUC19 plasmid is included to check the transformation efficiency of the One Shot® competent cells. Transform with 10 pg per 50 μL of cells using the protocols above and select for transformants on LB containing 50 μg/mL ampicillin.

Type of Cells Volume to Plate Expected Efficiency
TOP10 Chemically Competent10 μL + 20 μL S.O.C.1 × 109 cfu/μg DNA
Mach1™-T1R Chemically Competent10 μL + 20 μL S.O.C.1 × 109 cfu/μg DNA
OmniMAX™ 2-T1R Chemically Competent20 μL of a 1:50 dilution>5 × 109 cfu/μg DNA
TOP10 Electrocompetent20 μL of a 1:10 dilution>1 × 109 cfu/μg DNA



One Shot® Electroporation

We recommend electroporation over chemical transformation for higher transformation efficiency and for less bia  against large recombinant plasmids. Use ONLY electrocompetent cells for electroporation to avoid arcing.

  1. Add 2 μL of the TOPO® Cloning reaction to one vial of One Shot® electrocompetent cells and mix gently. Do not mix by pipetting up and down. Avoid formation of bubbles.

  2. Carefully transfer cells and DNA to a chilled 0.1 cm electroporation cuvette. Note: The volume of cells should be between 50 and 80 μL to reduce the chance of arcing.

  3. Electroporate your samples using your own protocol and your electroporator. Note: If you have problems arcing, see the next page.

  4. Immediately add 450 μL of room temperature S.O.C. medium and mix well.

  5. Transfer the solution to a 15 mL snap-cap tube (e.g. Falcon) and shake for at least 1 hour at 37°C to allow expression of the antibiotic resistance genes.

  6. Spread 20 to 150 μL from each transformation on a prewarmed LB plate containing 50 μg/mL kanamycin or 25 μg/mL Zeocin™. For the control reaction only, spread 50 μL each on a prewarmed LB plate containing 50 μg/mL kanamycin and on an LB plate containing 50 μg/mL kanamycin and 50 μg/mL ampicillin.

  7. Incubate plates overnight at 37°C.

  8. An efficient TOPO® Cloning reaction produces several hundred colonies. For the control transformation plates, see Analyzing the Control Transformation. To analyze clones containing your insert, see Analyzing Positive Clones


Addition of the Stop Solution in Step 3 of the TOPO® Cloning Reaction brings the final concentration of NaCl and MgCl2 in the TOPO® Cloning reaction to 50 mM and 10 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


One Shot® Chemical Transformation

Use this protocol only with One Shot® TOP10 chemically competent cells.

  1. Add 2 μL of the TOPO® Cloning reaction into a vial of One Shot® cells and mix gently. Do not mix by pipetting up and down.

  2. Incubate on ice for 30 minutes.

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

  4. Immediately transfer the tubes to ice and incubate for 2 minutes.

  5. Add 250 μL of room temperature S.O.C. medium.

  6. Cap the tube tightly and shake the tube horizontally at 37°C for 1 hour. Place on ice.

  7. Spread 50–150 μL from each transformation on a prewarmed plate. For the control transformation, spread 50 μL each on a prewarmed LB plate containing 50 μg/mL kanamycin and on an LB plate containing 50 μg/mL kanamycin and 50 μg/mL ampicillin.

  8. Incubate plates overnight at 37°C.

  9. An efficient TOPO® Cloning reaction produces several hundred colonies. For the control transformation plates, see Analyzing the Control Transformation, below. To analyze clones containing your insert, see Analyzing Positive Clones, below.


Note: Larger plasmids will produce fewer colonies.

Analyzing Transformants

Analyzing the Control Transformation

The control PCR product contains a functional ampicillin resistance gene and only recombinant colonies will grow on kanamycin and ampicillin. Dividing the total number of colonies that grow on kanamycin and ampicillin by the number of colonies that grow on the kanamycin only plate will give you a reasonable estimate of cloning efficiency. For chemically competent cells, >70% of these colonies will be ampicillin resistant; for electrocompetent cells, >80% will be ampicillin resistant.

Relatively few colonies will be produced in the vector-only reaction. Most of these will be frameshift mutations that disrupt the CcdB reading frame leading to the production of viable colonies.

Analyzing Positive Clones

  1. Pick ~10 colonies and culture them overnight in LB medium containing 50 μg/mL kanamycin. Alternatively, you may use Low Salt LB medium containing 25 μg/mL Zeocin™. Note:  If you transformed One Shot® Mach1™-T1R competent E. coli, you may inoculate overnight-grown colonies and culture them for 4 hours in prewarmed LB medium containing 50 μg/mL kanamycin or Low Salt LB medium containing 25 μg/mL Zeocin™ before isolating plasmid. For optimal results, we recommend inoculating as much of a single colony as possible.

  2. Isolate plasmid DNA using your method of choice. If you need ultra-pure plasmid DNA for automated or manual sequencing, we recommend the PureLink™ HQ Mini Plasmid Purification Kit.

  3. Analyze the plasmids by restriction analysis to confirm the presence and correct orientation of the insert. Use a restriction enzyme or a combination of enzymes that cut once in the vector and once in the insert. Sequencing You may sequence your construct to confirm that your gene is cloned in the correct orientation. The M13 Forward (–20) and M13 Reverse primers are included to help you sequence your insert.

Sequencing

You may sequence your construct to confirm that your gene is cloned in the correct orientation. The M13 Forward (–20) and M13 Reverse primers are included to help you sequence your insert.

Alternative Method of Analysis

You may wish to use PCR to directly analyze positive transformants. For PCR primers, use either the M13 Forward (–20) or the M13 Reverse primer and a primer that hybridizes within your insert. If you are using this technique for the first time, we recommend performing restriction analysis in parallel. Artifacts may be obtained because of mispriming or contaminating template. The protocol is provided below for your convenience. Other protocols are suitable. You may wish to use PCR to directly analyze positive transformants. For PCR primers, use either the M1  Forward (–20) or the M13 Reverse primer and a primer that hybridizes within your insert. If you are using this technique for the first time, we recommend performing restriction analysis in parallel. Artifacts may be obtained because of mispriming or contaminating template. The protocol is provided below for your convenience. Other protocols are suitable.

Materials Needed

PCR SuperMix High Fidelity (Cat. no. 10790-020) Appropriate forward and reverse PCR primers (20 μM each)

Procedure

  1. For each sample, aliquot 48 μL of PCR SuperMix High Fidelity into a 0.5 mL microcentrifuge tube. Add 1 μL each of the forward and reverse PCR primer.

  2. Pick 10 colonies and resuspend them individually in 50 μL of the PCR cocktail from Step 1, above. 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.

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

  6. Visualize by agarose gel electrophoresis.

Long-Term Storage

  1. Once you have identified the correct clone, purify the colony and make a glycerol stock for long term storage.

  2. Streak the original colony out for single colonies on LB plates containing 50 μg/mL kanamycin or 25 μg/mL Zeocin™.

  3. Isolate a single colony and inoculate into 1–2 mL of LB containing 50 μg/mL kanamycin or 25 μg/mL Zeocin™.

  4. Grow until culture is at mid-log phase (between 0.6 and 1.0 OD600).

  5. Mix 0.85 mL of culture with 0.15 mL of sterile glycerol and transfer to a cryovial.

  6. Store at –80°C.
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Troubleshooting

TOPO® Cloning

The table below lists some potential problems and possible solutions to help you troubleshoot the TOPO® Cloning procedure. We recommend performing the control reaction on parallel with your samples to help you evaluate your results.

Problem Solution
Incubating ligation reaction longer than 5 minutesBe sure to incubate for only 5 minutes for the ligation reaction. Incubations longer than 5 minutes will decrease transformation and cloning efficiencies.
Low yield of transformantsRemember to use the 6X TOPO® Cloning Stop Solution.
Incomplete extension during PCRBe sure to include a final extension step of 7 to 30 minutes during PCR. Longer PCR products will need a longer extension time.
Dilute PCR productYou will need >2 ng/μL PCR product. Concentrate the amount of PCR product by ethanol precipitation. Or produce more product and concentrate by ethanol precipitation.
PCR cloning artifacts ("false positives")TOPO® Cloning is very efficient for small fragments (<100 bp) present in certain PCR reactions. It is imperative that you gel-purify your PCR product
PCR product does not contain sufficient 3´ A-overhangs even though Taq polymerase is present in the polymerase miTaq 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).
Using ethidium bromide agarose gel electrophoresis to gel-purify PCR productIntercalation of ethidium bromide into the DNA and subsequent exposure to UV light will damage your DNA leading to lower cloning efficiencies. Use the crystal violet reagents in the kit to gel-purify your PCR product.


Gel Purification

The table below lists some potential problems and possible solutions to help you troubleshoot the gel purification procedure. We recommend performing the control reaction in parallel with your samples to help you evaluate your results.

Problem Solution
Low yield of PCR product on gelLoad more PCR product. You may have to optimize PCR conditions to obtain >200 ng.
Low yield of PCR product from gel purificationBe sure to melt gel slices thoroughly to ensure release of PCR product.
 Be sure to use TAE to prepare the crystal violet agarose gel. TBE will interfere with isolation of the PCR product with sodium iodide..
 Fragment may fail to elute from the S.N.A.P™ column. Warm TE buffer to 37°C and try eluting again.

Recipes

LB (Luria-Bertani) Medium and Plates

Do not use Luria-Bertani Medium with Zeocin™. See Low Salt LB medium below.


Composition:

1.0% Tryptone
0.5% Yeast Extract
1.0% NaCl
pH 7.0

  1. For 1 liter, dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 mL deionized water.

  2. Adjust the pH of the solution to 7.0 with NaOH and bring the volume up to 1 liter.

  3. Autoclave on liquid cycle for 20 minutes at 15 psi. Allow solution to cool to 55°C and add antibiotic (50 μg/mL of kanamycin) if needed.

  4. Store at room temperature or at 4°C.

LB agar plates

  1. Prepare LB medium as above, but add 15 g/L agar before autoclaving.

  2. Autoclave on liquid cycle for 20 minutes at 15 psi.

  3. After autoclaving, cool to ~55°C, add antibiotic (50 μg/mL of kanamycin), and pour into 10 cm plates.

  4. Let harden, then invert and store at 4°C, in the dark.

Low Salt LB Medium

Reduce the salt in LB medium if you are using Zeocin™ for selection.

Composition:

1.0% Tryptone
0.5% Yeast Extract
0.5% NaCl
pH 7.5

  1. For 1 liter, dissolve 10 g tryptone, 5 g yeast extract, and 5 g NaCl in 950 mL deionized water. For plates, be sure to add 15 g/l agar.

  2. Adjust the pH of the solution to 7.5 with NaOH and bring the volume up to 1 liter.

  3. Autoclave on liquid cycle for 20 minutes at 15 psi. Allow solution to cool to 55°C and add Zeocin™ to a final concentration of 25 μg/mL.

  4. Store at room temperature or at 4°C.
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References

  1. 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).

  2. Bernard, P., Gabant, P., Bahassi, E. M., and Couturier, M. (1994). Positive Selection Vectors Using the F Plasmid ccdB Killer Gene. Gene 148, 71-74.

  3. 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.

  4. Rand, K. N. (1996). Crystal Violet Can Be Used to Visualize DNA Bands During Gel Electrophoresis and to Improve Cloning Efficiency. Elsevier Trends Journals Technical Tips Online, T40022.

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

  6. Shuman, S. (1994). Novel Approach to Molecular Cloning and Polynucleotide Synthesis Using Vaccinia DNA Topoisomerase. J. Biol. Chem. 269, 32678-32684.
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MAN0001691       Rev. date: 20-May-2010