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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.
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.
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 TOPOCloning 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.
The flow chart below outlines the experimental steps necessary to clone your blunt-end PCR product
This kit is specifically designed to clone blunt-end PCR products generated by thermostable proofreading polymerases such as Invitrogen™ Platinum™ SuperFi™ 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.
You will need the following reagents and equipment for PCR.
Note: dNTPs (adjusted to pH 8) are provided in the kit.
Set up a 25 µl or 50 µl PCR reaction using the guidelines below:
After cycling, place the tube on ice or store at -20ºC for up to 2 weeks. Proceed to Checking the PCR Product, below.
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.
Invitrogen Platinum SuperFi DNA Polymerase is a proofreading DNA polymerase that combines exceptional fidelity with trusted Platinum hot-start technology. Featuring >100x the fidelity of Taq polymerase, Platinum SuperFi DNA Polymerase is ideally suited for cloning, mutagenesis, and other applications that benefit from sequence accuracy.
Benefits of Platinum SuperFi DNA Polymerase include:
Platinum SuperFi DNA Polymerase is engineered with a DNA-binding domain, resulting in high processivity and increased resistance to PCR inhibitors. This feature also enables fast-cycling protocols and amplification of long targets. The Platinum hot-start technology is based on proprietary antibodies that inhibit enzyme activity until the initial PCR denaturation step, preventing nonspecific amplification and primer degradation.
Platinum SuperFi DNA Polymerase is supplied with a separate vial of Invitrogen SuperFi GC Enhancer designed for GC-rich templates (>65% GC).
The following procedure is suggested as a starting point when using Platinum SuperFi DNA Polymerase in PCR amplification.
1Important! Always use the Tm calculator on our website at thermofisher.com/tmcalculator to calculate the Tm of your primers and the recommended annealing temperature.
Note: Consider the volumes for all components listed in steps 2 and 4 to determine the correct amount of water required to reach your final reaction volume.
Component | 50-μL rxn | Final conc. |
---|---|---|
Water, nuclease-free | to 50 μL | |
5X SuperFi Buffer1 | 10 μL | 1X |
10 mM dNTP mix | 1 μL | 0.2 mM each |
5X Super GC Enhancer (optional) 2 | 10 μL | 1X |
Platinum SuperFi DNA Polymerase | 0.5 μL | 0.02 U/μL |
Component | 50-μL rxn | Final conc. |
---|---|---|
10 μM forward primer | 2.5 μL | 0.5 μM |
10 μM reverse primer | 2.5 μL | 0.5 μM |
Template DNA1 | varies | varies |
1 Optimal amount of low complexity DNA (plasmid, phage, BAC DNA) is 1 pg–10 ng per 50 μL reaction, but it can be varied from 0.1 pg to 50 ng per 50 μL reaction. Optimal amount of genomic DNA is 5–50 ng per 50 μL reaction, but it can be varied from 0.1 ng to 250 ng per 50 μL reaction.
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.
The S.N.A.P. MiniPrep Kit 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.
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.
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.
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.
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.
Once you have produced the desired PCR product, you are ready to TOPOClone 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.
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.
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.
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.
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.
In addition to general microbiological supplies (e.g. plates, spreaders), you will need the following reagents and equipment.
For each transformation, you will need one vial of competent cells and two selective plates.
If you are transforming One ShotMach1-T1R Chemically Competent E. coli, it is essential that selective plates are prewarmed to 37° prior to spreading for optimal growth of cells.
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:
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.
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.
PCR SuperMix High Fidelity or equivalent. Appropriate forward and reverse PCR primers (20 µM each)
Once you have identified the correct clone, be sure to prepare a glycerol stock for long-term storage.
For Research Use Only. Not for use in diagnostic procedures.