Introduction

Zero Blunt® TOPO® PCR Cloning provides a highly efficient, 5 minute, one-step cloning strategy ("TOPO® Cloning") for the direct insertion of blunt-end PCR products into a plasmid vector. No ligase, post-PCR procedures, or PCR primers containing specific sequences are required.
 
How Topoisomerase I Works

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.
 
TOPO® Cloning

The plasmid vector (pCR®-Blunt II-TOPO®) is supplied linearized with Vaccinia virus DNA topoisomerase I covalently bound to the 3´ end of each DNA strand (referred to as "TOPO®-activated" vector). The TOPO®Cloning Reaction can be transformed into chemically competent cells or electroporated directly into electrocompetent cells.
 
In addition, pCR®-Blunt II-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 LacZa fragment. Ligation of a blunt-end PCR product disrupts expression of the lacZa-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.
 




Experimental Outline

The flow chart below outlines the experimental steps necessary to clone your blunt-end PCR product


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Producing Blunt-End PCR Products

Introduction

This kit is specifically designed to clone blunt-end PCR products generated by thermostable proofreading polymerases such as Platinum® Pfx DNA Polymerase. Follow the guidelines below to produce your blunt-end PCR product. 
 
Do not add 5´ phosphates to your primers for PCR. The PCR product synthesized will not ligate into pCR®-Blunt II-TOPO®.

 
Materials Supplied by the User

You will need the following reagents and equipment for PCR.
Note:   dNTPs (adjusted to pH 8) are provided in the kit.

  • Thermostable proofreading polymerase
  • 10X PCR buffer appropriate for your polymerase
  • Thermocycler
  • DNA template and primers for your PCR product 


Producing PCR Products

Set up a 25 µl or 50 µl PCR reaction using the guidelines below:

  • Follow the instructions and recommendations provided by the manufacturer of your thermostable, proofreading polymerase to produce blunt-end 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. 

Checking the PCR Product

After you have produced your blunt-end PCR product, use agarose gel electrophoresis to verify the quality and quantity of your PCR product. Check for the following outcomes below.
 
Be sure you have a single, discrete band of the correct size. If you do not have a single, discrete band, follow the manufacturer’s recommendations for optimizing your PCR with the polymerase of your choice. Alternatively, you may gel-purify the desired product.  
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Protocol: Generating PCR Products with Pfx™50 DNA Polymerase

Introduction
 
Pfx50™ DNA Polymerase is a fusion enzyme consisting of recombinant DNA polymerase from the archaean Thermococcus zilligii fused to an accessory protein. The highly thermostable polymerase possesses a proofreading 3’ → 5’ exonuclease activity, while the accessory protein stabilizes primer-template complexes in PCR.
 
Pfx50™ DNA Polymerase offers 50 times better fidelity than Taq DNA polymerase,coupled with high specificity and an extremely fast elongation rate (as fast as 15 seconds per kb). In addition, the fusion enzyme has an intrinsic hot-start capability for room-temperature reaction assembly.
 
Note:

  • 10X Pfx50™ PCR buffer contains BSA; store at –20ºC.
  • Pfx50™ DNA Polymerase produces blunt-end PCR products, which can be used with Directional TOPO® Cloning and Zero Blunt® TOPO® Cloning technologies. 


Component 100 Rxn Kit 500 Rxn Kit
Pfx50™ DNA Polymerase (5 U/µl)100 µl 500 µl
10X Pfx50™ PCR Mix 1.3 ml 2 × 1.3 ml
50-mM Magnesium Sulfate1 ml1 ml
 
Pfx50™ DNA Polymerase Storage Buffer

20 mM Tris-HCl (pH 8.0), 40 mM KCl, 0.1 mM EDTA, 1 mM DTT, stabilizers, and 50% (v/v) glycerol 

Unit Definition

One unit of Pfx50™ DNA Polymerase incorporates 10 nmol of deoxyribonucleotide into acid-insoluble material in 30 min at 74°C.
 
General Recommendations and Guidelines for PCR

General PCR parameters and troubleshooting information are documented in Innis, et al.
 
Template:  Pfx50™ DNA Polymerase is suitable for amplifying targets up to 4 kb from the following templates:
Template                    Amount
Genomic DNA            1–200 ng
Plasmid DNA             1–100 pg
cDNA                        3­–5 µl from 10 ng to 1 µg starting total RNA

Amplification of longer targets (up to 7 kb) may be possible, but may require more template and longer elongation times.
 
Primers:  Use 0.3 µM per primer as a general starting point. For larger amounts of template (e.g., 200 ng genomic DNA), increasing the concentration up to 0.5 µM per primer may improve yield.
 
Annealing Temperature:  The annealing temperature is slightly higher than with typical PCR. The optimal annealing temperature should be ~2ºC lower than the Tm of the primers used. A range of 60–68ºC is recommended.
 
MgSO4 MgSO4 is included in the 10X Pfx50™ PCR Mix at a final concentration of 1.2 mM, which is sufficient for most templates. For further optimization, add 0.1 µl to 1.0 µl of 50-mM MgSO4.
 
Extension Time:  As little as 15 seconds per kb may be used; 30 seconds per kb is suitable for most targets. Use up to 60 seconds per kb for maximum yield.
 
Protocol

 
The following procedure is suggested as a starting point when using Pfx50™ DNA Polymerase in any PCR amplification.

  1. Program the thermal cycler as follows (see the note on annealing temperature above):
     
    Initial denaturation:      94ºC for 2 minutes
    35 cycles of:
    Denaturation:              94ºC for 15 seconds
    Annealing:                  60–68ºC (Tm of primers minus 2ºC) for 10–30 seconds
    Extension:                  68ºC for 30–60 seconds per kb of PCR product
    Final extension:          68ºC for 5 minutes
     

  2. Add the following components to an autoclaved microcentrifuge tube at room temperature (for multiple reactions, prepare a Master Mix of common components to enable accurate pipetting):

    Component Volume Final Conc.
    10X Pfx50™ PCR Mix
    5 µl   1X 
    10 mM dNTP Mix 1.5 µl 0.3 mM each
     Primer mix (10 µM each)1.5 µl0.3 µM each
    Template DNA ≥1 µl
    As required

     Pfx50™ DNA Polymerase (5 U/µl)
     1 µl
    5 units
    Autoclaved, distilled water
    50 ul


     
  3. Cap the tube, tap gently to mix, and centrifuge briefly to collect the contents.

  4. Place the tube in the thermal cycler and run the program from Step 1. After cycling, maintain the reaction at 4ºC. Samples can be stored at  –20ºC until use.
Analyze products using E-Gel® Pre-Cast agarose gels or standard agarose gel electrophoresis. Visualize by staining with SYBR Safe™ DNA gel stain or ethidium bromide.
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Purifying PCR Products

Introduction

Smearing, multiple banding, primer-dimer artifacts, or large PCR products (>1 kb) may necessitate gel purification. If you intend to purify your PCR product, be extremely careful to remove all sources of nuclease contamination. There are many protocols to isolate DNA fragments or remove oligonucleotides. Refer to Current Protocols in Molecular Biology, Unit 2.6 (Ausubel et al., 1994) for the most common protocols. Three simple protocols are provided below.
 
Using the S.N.A.P.™ MiniPrep Kit

The S.N.A.P.™ MiniPrep Kit (Catalog no. K1900-01) allows you to rapidly purify PCR products from regular agarose gels. You will need to prepare 6 M sodium iodide, 10 mM sodium sulfite in sterile water before starting. Sodium sulfite prevents oxidation of NaI.
 
  1. Electrophorese amplification reaction on a 1 to 5% regular agarose gel using TAE buffer. Do not use TBE buffer. It will interfere with DNA binding to the S.N.A.P.™ resin 
  2. Cut out the gel slice containing the PCR product and melt it at 65°C in 2 volumes of 6 M NaI. Minimize exposure to UV to prevent damage to your DNA.
  3. Add 1.5 volumes Binding Buffer (provided in the S.N.A.P.™ MiniPrep Kit).
  4. Load solution (no more than 1 ml at a time) from Step 3 onto a S.N.A.P.™ column. Centrifuge 1 minute at full speed in a microcentrifuge and discard the supernatant.
  5. If you have solution remaining from Step 3, repeat Step 4.
  6. Add 900 µl of the Final Wash Buffer (provided in the S.N.A.P.™ MiniPrep Kit).
  7. Centrifuge 1 minute at full speed in a microcentrifuge and discard the supernatant. Repeat.
 
To elute the purified PCR product, transfer the column to a sterile microcentrifuge tube and add 40 µl of TE or sterile water.
 
Centrifuge at full speed for 30 seconds. The DNA will be eluted into the microcentrifuge tube. Use 4 µl for the TOPO® Cloning reaction and proceed as described. 

Quick S.N.A.P.™ Method

An easier method is to simply cut out the gel slice containing your PCR product, place it on top of the S.N.A.P.™ column bed, and centrifuge at full speed for 10 seconds. Use 1-2 µl of the flow-through in the TOPO® Cloning reaction. Be sure to make the gel slice as small as possible for best results. 

Low-Melt Agarose

Method

Note that gel purification will result in a dilution of your PCR product and a less efficient TOPO® Cloning reaction. Use only chemically competent cells for transformation.
 
  1. Electrophorese as much as possible of your PCR reaction on a low-melt agarose gel (0.8 to 1.2%) in TAE buffer.
  2. Visualize the band of interest and excise the band. Minimize exposure to UV to prevent damage to your DNA.
  3. Place the gel slice in a microcentrifuge tube and incubate the tube at 65°C until the gel slice melts.
  4. Place the tube at 37°C to keep the agarose melted.
  5. Use 4 µl of the melted agarose containing your PCR product in the TOPO® Cloning reaction. 
  6. Incubate the TOPO® Cloning reaction at 37°C for 5 to 10 minutes. This is to keep the agarose melted.
  7. Transform 2 to 4 µl directly into One Shot® competent E. coli using the protocols described. 
 
Note that the cloning efficiency may decrease with purification of the PCR product. You may wish to optimize your PCR to produce a single band 

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Performing the TOPO® Cloning Reaction

Introduction

Once you have produced the desired PCR product, you are ready to TOPO® Clone it into the pCR® -Blunt II-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 before beginning. 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.
 
Using Salt Solution in the TOPO® Cloning Reaction

You will perform TOPO® Cloning in a reaction buffer containing salt (i.e., using the stock salt solution provided in the kit). Note that the amount of salt added to the TOPO® Cloning reaction varies depending on whether you plan to transform chemically competent cells or electrocompetent cells.

  • If you are transforming chemically competent E. coli, use the stock Salt Solution as supplied and set up the TOPO® Cloning reaction as directed below.
  • If you are transforming electrocompetent E. coli, the amount of salt in the TOPO® Cloning reaction must be reduced to 50 mM NaCl, 2.5 mM MgCl2 to prevent arcing during electroporation. Dilute the stock Salt Solution 4-fold with water to prepare a 300 mM NaCl, 15 mM MgCl2 Dilute Salt Solution. Use the Dilute Salt Solution to set up the TOPO® Cloning reaction as directed.


Performing the TOPO® Cloning Reaction

Use the procedure below to perform the TOPO® Cloning reaction. Set up the TOPO® Cloning reaction using the reagents in the order shown, and depending on whether you plan to transform chemically competent E. coli or electrocompetent E. coli.

  • Note:   The blue color of the pCR® II-Blunt-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 (1:4)
--
1 µl
Sterile Water
add to a final volume of 5 µl
add to a final volume of 5 µl
pCR®II-Blunt-TOPO®
1 µl
1 µl
Final Volume
6 µl
6 µl


*Store all reagents at -20°C when finished. Salt solutions and water can be stored at room temperature or +4°C. 
 

  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 Transforming One Shot® Competent Cells.

 

  • Note:  You may store the TOPO® Cloning reaction at -20°C overnight. 
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Transforming One Shot® Competent Cells

Introduction

Once you have performed the TOPO® Cloning reaction, you will transform your pCR®-Blunt II-TOPO® construct into competent E. coli provided with your kit. Protocols to transform chemically competent and electrocompetent E. coli are provided below.
 
Materials Supplied by the User

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

  • TOPO®Cloning reaction from Performing the TOPO® Cloning Reaction, Step 2
  • S.O.C. medium (included with the kit)
  • 42°C water bath or electroporator with 0.1 cm cuvettes
  • 15 ml snap-cap plastic tubes (sterile) (electroporation only)
  • LB plates containing 50 µg/ml kanamycin or Low Salt LB plates containing 25 µg/ml Zeocin™ (use two plates per transformation; see recipes)
  • 37°C shaking and non-shaking incubator 

Preparing for Transformation

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

  • Equilibrate a water bath to 42°C or set up electroporator
  • Bring the vial of S.O.C. medium to room temperature.
  • Warm LB plates containing 50 µg/ml kanamycin or 25 µg/ml Zeocin™ at 37°C for 30 minutes.
  • Thaw on ice 1 vial of One Shot® cells for each transformation.
  
If you are transforming One Shot®Mach1™-T1R Chemically Competent E. coli, it is essential that selective plates are prewarmed to 37° prior to spreading for optimal growth of cells. 
 
One Shot® Chemical Transformation

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

  2. Incubate on ice for 5 to 30 minutes.

  3. Note:  
    Longer incubations on ice do not seem to have any affect on transformation efficiency. The length of the incubation is at the user’s discretion.

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

  5. Immediately transfer the tubes to ice.

  6. Add 250 µl of room temperature S.O.C. medium.

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

  8. 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 S.O.C. medium. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies. Incubate plates over night at 37°C.

  9. An efficient TOPO® Cloning reaction will produce several hundred colonies. Pick ~10 colonies for analysis (see Analyzing Transformants).
 
One Shot® Electroporation

  1. Add 2 µl of the TOPO® Cloning reaction to a vial (50 µl) of One Shot® Electrocomp™ E. coli 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 cuvette.

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

  4. Immediately add 250 µl of room temperature S.O.C. medium to the cuvette.

  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 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 S.O.C. medium. We recommend that you plate two different volumes to ensure that at least one plate will have well-spaced colonies. Incubates plates over night at 37°C.

  7. An efficient TOPO® Cloning reaction will produce several hundred colonies. Pick ~10 colonies for analysis.
 
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
  • Precipitate TOPO® Cloning reaction and resuspend in water prior to electroporation 
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Analyzing Transformants

Analyzing Positive Clones

  1. Take the 10 colonies and culture them overnight in LB medium containing 50 µg/ml kanamycin or 25 µg/ml Zeocin™. Be sure to save the original colony by patching to a fresh plate, if needed.
  2.  
    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 25 µg/ml Zeocin™ before isolating plasmid. For optimal results, we recommend inoculating as much of a single colony as possible.
     
     
  3. 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 (Catalog no. K2100-01).

  4. 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. For the full sequence of pCR®-Blunt II-TOPO®, refer to our Web site (www.invitrogen.com). ) or contact Technical Service.
 
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.
 
Materials Needed

PCR SuperMix High Fidelity (Invitrogen, Catalog no. 10790-020) or equivalent. 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

Once you have identified the correct clone, be sure to prepare a glycerol stock for long-term storage.
 
  1. Streak the original colony out on LB plates containing 50 µg/ml kanamycin (or 25 µg/ml Zeocin™)
  2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50 µg/ml kanamycin (or 25 µg/ml Zeocin™).
  3. Grow with shaking to log phase (OD600 = ~0.5)
  4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial. 
  5. Store at -80°C. 
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Reference

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