Cloning of Taq polymerase-amplified PCR products

TOPO TA Cloning 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.

How It Works

The plasmid vector (pCRII-TOPO or pCR 2.1-TOPO) is supplied linearized with:

• Single 3´-thymidine (T) overhangs for TA Cloning
• Topoisomerase I covalently bound to the vector (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).

Experimental Outline

• Produce Your PCR Product
• Set Up TOPO Cloning Reaction (Mix Together PCR Product and TOPO Vector)
• Incubate 5 Minutes at Room Temperature
• Transform TOPO Cloning Reaction into One Shot Competent Cells
• Select and Analyze 10 White or Light Blue Colonies for Insert

Introduction

It is important to properly design your PCR primers to ensure that you obtain the product you need for your studies. Once you have decided on a PCR strategy and have synthesized the primers, you are ready to produce your PCR product. Remember that your PCR product will have single 3´ adenine overhangs.
Do not add 5´ phosphates to your primers for PCR. The PCR product synthesized will not ligate into pCR 2.1-TOPO or pCRII-TOPO.

Materials Supplied by the User

You will need the following reagents and equipment:

• Taq polymerase or a polymerase mixture including Taq polymerase (e.g., Platinum Taq DNA Polymerase High Fidelity
• Thermocycler
• DNA template and primers for PCR product

Producing PCR Products Using Taq Polymerase

1. Set up the following 50-µl PCR reaction using Taq DNA polymerase. 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.




DNA Template	10-100 ng
10X PCR Buffer	5 µl
50 mM dNTPs	0.5 µl
Primers (100-200 ng each)	1 µM each
Water	add to a final volume of 49 µl
Taq Polymerase (1 unit/µl)	1 µl
Total Volume	50 µl



2. Check the PCR product by agarose gel electrophoresis. You should see a single, discrete band. If you do not see a single band, 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 TOPO TA Cloning Kit. Take special care to avoid sources of nuclease contamination. Alternatively, you may optimize your PCR to eliminate multiple bands and smearing (Innis et al., 1990).

Introduction

Platinum Taq  DNA Polymerase High Fidelity is an enzyme mixture composed of recombinant Taq  DNA polymerase, Pyrococcus species GB-D polymerase, and Platinum Taq Antibody.  Pyrococcus species GB-D polymerase possesses a proofreading ability by virtue of its 3´ to 5´ exonuclease activity.  Mixture of the proofreading enzyme with Taq  DNA polymerase increases fidelity approximately six times over that of Taq  DNA polymerase alone and allows amplification of simple and complex DNA templates over a large range of target sizes. Targets 12–20 kb can be amplified with some optimization. Targets greater than 20 kb require thorough optimization. The enzyme mixture is provided with an optimized buffer that improves enzyme fidelity and amplification of difficult templates.
 
An anti-Taq  DNA polymerase antibody complexes with and inhibits polymerase activity.  Activity is restored after the denaturation step in PCR cycling at 94°C, thereby providing an automatic “hot start” for Taq DNA polymerase in PCR.  Hot starts in PCR provide increased sensitivity, specificity, and yield, while allowing assembly of reactions at room temperature. The use of this antibody helps reduce PCR optimization requirements, reaction set-up time and effort, handling of reaction components, and contamination risk, thereby improving PCR results.
 
Platinum Taq  DNA Polymerase High Fidelity is supplied at the same 5 unit per µl concentration as Platinum Taq  DNA Polymerase.  No modification to PCR reactions or protocols are necessary.

Platinum Taq DNA Polymerase High Fidelity Storage Buffer

20 mM Tris-HCl (pH 8.0), 40 mM NaCl, 2 mM Sodium Phosphate, 0.1 mM EDTA, 1 mM DTT, stabilizers, and 50% (v/v) glycerol

10X High Fidelity PCR Buffer

600 mM Tris-SO4 (pH 8.9), 180 mM Ammonium Sulfate

Unit Definition

One unit of Platinum Taq DNA Polymerase High Fidelity incorporates 10 nmol of deoxyribonucleotide into acid-precipitable material in 30 min at 74°C.

PCR Precautions

An extension temperature of 68°C, the use of thin-walled reaction tubes for targets above 5 kb, the use of one unit of enzyme for targets above 12 kb, and an increased primer concentration of 0.4 µM for plasmid and lambda targets are all recommended conditions.

Protocol

The following procedure is suggested as a guideline and starting point when using Platinum Taq  DNA Polymerase High Fidelity in any PCR amplification. Reaction size may be altered to suit user preferences. 

1. Add the following components to a autoclaved microcentrifuge tube at either ambient temperature, or on ice:
 

Components	Volume	Final Concentration
10X High Fidelity PCR Buffer	5 µl	1X
10 mM dNTP mixture	1 µl	0.2 mM each
50 mM MgSO4	2 µl	2 mM
Primer mix (10 µM each)	1 µl	0.2 µM each
Template DNA	> 1 µl	(as required)
Platinum Taq High Fidelity	0.2 µl	1.0 unit*
Autoclaved, distilled water	to 50 µl	Not applicable



2. Mix contents of the tubes and overlay with mineral or silicone oil, if necessary.
3. Cap the tubes and centrifuge briefly to collect the contents.
4. Incubate tubes in a thermal cycler at 94°C for 30 s to 2 min to completely denature the template and activate the enzyme.  Do not denaturate for more than 30 s if target is greater than 12 kb
5. Perform 25-35 cycles of PCR amplification as follows:
    
                      Denature       94°C for 15–30 s
                      Anneal          55°C for 15–30 s
                      Extend           68°C for 1 min per kb


6. Maintain the reaction at 4°C after cycling.  The samples can be stored at –20°C until use.
7. Analyze the products by agarose gel electrophoresis and visualize by ethidium bromide staining. Use appropriate molecular weight standards.
   
   
   TOP

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. Two simple protocols are provided below for your convenience.

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 solution in sterile water before starting. Sodium sulfite prevents oxidation of NaI.

1. Electrophorese amplification reaction on a 1 to 5% regular TAE agarose gel.
2. Cut out the gel slice containing the PCR product and melt it at 65°C in 2 volumes of 6 M NaI.
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.
8. Elute the purified PCR product in 40 µl of TE or water. Use 4 µl for the TOPO Cloning reaction.

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.
 
   1. Electrophorese all of your PCR reaction on a low-melt TAE agarose gel (0.8 to 1.2%).
   2. Visualize the band of interest and excise the band.
   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 chemically competent cells.  
 
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.

Introduction

Once you have produced the desired PCR product, you are ready to TOPO Clone it into the pCR 2.1-TOPO or pCR 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 sections detailing transformation of competent cells(pages 6-10) before beginning. If this is the first time you have TOPO Cloned, perform the control reactions on pages 17-18 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. 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 (see below).  For this reason two different TOPO Cloning reactions are provided to help you obtain the best possible results. Read the following information carefully.

Transforming 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.

Transforming 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.  

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 or electrocompetent TOP10 or chemically competent DH5a™-T1R, Mach1™-T1R, or TOP10F' One Shot E. coli. Additional information on optimizing the TOPO Cloning reaction for your needs can be found below.
 
Note:  The red color of the TOPO vector solution is normal and is used to visualize the solution.  

*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

   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 General Guidelines for Transforming Competent Cells.
 
Note:  You may store the TOPO Cloning reaction at -20°C overnight.

Introduction

Once you have performed the TOPO Cloning reaction, you will transform your pCR2.1-TOPO or pCR II-TOPO construct into competent E. coli provided with your kit. General guidelines for transformation are provided below. For transformation protocols, refer to specific information provided with your competent cells. Transforming One Shot Mach1-T1R Competent Cells or Transforming One Shot DH5a-T1R, TOP10, and TOP10F´ Competent Cells depending on the competent E. coli you wish to transform.

Selecting a One Shot Chemical Transformation Protocol

Two protocols are provided to transform One Shot  chemically competent E. coli. Consider the following factors when choosing the protocol that best suits your needs.

If you will be using kanamycin as the selective agent for chemical transformation, use the regular chemical transformation protocol. The rapid chemical transformation protocol is only suitable for transformations using ampicillin selection.
If you use a plasmid template for your PCR that carries either the ampicillin or kanamycin resistance marker, we recommend that you use the other selection agent to select for transformants. For example, if the plasmid template contains the ampicillin resistance marker, then use kanamycin to select for transformants. The template is carried over into the TOPO Cloning and transformation reactions, resulting in transformants that are ampicillin resistant and white, but are not the desired construct.

Analyzing Positive Clones

1. Culture 10 white or light blue colonies overnight in LB medium containing 50 µg/ml ampicillin or 50 µg/ml kanamycin.

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 ampicillin or 50 µg/ml kanamycin 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 S.N.A.P. MiniPrep Kit (Catalog no. K1900-01) or the S.N.A.P. MidiPrep Kit (Catalog no. K1910-01).
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. Refer to the vector maps (pCR 2.1-TOPO or pCR II-TOPO) for the sequence surrounding the TOPO TA Cloning site. For the full sequence of either vector, refer to www.thermofisher.com or contact Technical Service.

Analyzing Transformants by PCR

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)
• Appropriate forward and reverse PCR primers (20 µM each)

Procedure

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.

1. 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.
2. Incubate the reaction for 10 minutes at 94°C to lyse the cells and inactivate nucleases.
3. Amplify for 20 to 30 cycles.
4. For the final extension, incubate at 72°C for 10 minutes. Store at +4°C.
5. 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. We recommend that you store a stock of plasmid DNA at -20°C.
 
   1. Streak the original colony out on LB plates containing 50 µg/ml ampicillin or 50 µg/ml kanamycin. 
   2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50 µg/ml ampicillin or kanamycin.
   3. Grow until culture reaches stationary phase.
   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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