Cloning of A-tailed PCR fragments using conventional ligase method

The TA Cloning^® Kit with pCR^®2.1 provides a quick, one-step cloning strategy for the direct insertion of a PCR product into a plasmid vector.
 
Advantages

Using the TA Cloning^® Kit:

• Eliminates any enzymatic modifications of the PCR product
• Does not require the use of PCR primers that contain restriction sites

 
How TA Cloning^® Works

Taq polymerase has a non template-dependent activity that adds a single deoxyadenosine (A) to the 3´ ends of PCR products. The linearized vector supplied in this kit has single 3´ deoxythymidine (T) residues. This allows PCR inserts to ligate efficiently with the vector.
 
Diagram

The diagram below shows the concept behind the TA Cloning^® method.




Thermostable polymerases containing extensive 3´ to 5´ exonuclease activity, such as Platinum^® Pfx, do not leave 3´ A-overhangs. PCR products generated with Taq polymerase have a high efficiency of cloning in the TA Cloning^® system as the 3´ A-overhangs are not removed. However, if you use a proofreading polymerase or wish to clone blunt-ended fragments, you can add 3´ A-overhangs by incubation with Taq at the end of your cycling program. 

Alternatively, you may want to try the Zero Blunt^® PCR Cloning Kit (Catalog nos. K2700-20 and K2750-20). This kit offers efficient cloning of blunt-end PCR products generated using thermostable, proofreading polymerases. For more information, visit our website or contact Technical Service.

Storage Instructions

The TA Cloning^® Kits are shipped on dry ice and contain a box of TA Cloning^® Reagents (Box 1) and a box of One Shot^® Competent Cells (Box 2). Catalog nos. K2020-20 and K2020-40 are not supplied with One Shot^® Competent Cells. Store Box 1 at -20°C in a non-frost-free freezer and Box 2 at -80°C.
 
TA Cloning^® Reagents

TA Cloning^® Reagents (Box 1) are listed below. Note that the user must supply Taq Polymerase. Forty reaction kits are supplied as two 20 reaction kits. Store Box 1 at -20ºC.

One Shot^® Reagents

The table below describes the items included in the One Shot^® competent cell kit. Catalog nos. K2020-20 and K2020-40 are not supplied with competent cells. Forty reaction kits are supplied as two 20 reaction kits.
The transformation efficiency for TOP10F´ and TOP10 cells is 1 x 10⁹ cfu/µg DNA. The transformation efficiency for INVaF´ is 1 x 10⁸ cfu/µg DNA. Store competent cells at -80ºC.

Genotype of INVaF´

F´ endA1 recA1 hsdR17 (rk^- , mk⁺ ) supE44 thi-1 gyrA96 relA1 f80lacZD M15 D (lacZYA-argF)U169 l-
 
Genotype of TOP10F´

F´ [lacIq Tn10 (TetR)] mcrA D (mrr-hsdRMS-mcrBC) F80lacZD M15 D lacC74 recA1 araD139 D(ara-leu)7697 galU galK rpsL (Str^R ) endA1 nupG
 
Genotype of TOP10

F- mcrA D (mrr-hsdRMS-mcrBC) F80lacZDM15 D lacC74 recA1 araD139 D (ara-leu)7697 galU galK rpsL (Str^R ) endA1 nupG

Introduction

To clone your gene of interest into pCR^® 2.1, you must first generate a PCR product. The PCR product is ligated into pCR^® 2.1 and transformed into competent cells. Since the PCR product can ligate into the vector in either orientation, individual recombinant plasmids need to be analyzed to confirm proper orientation. The correct recombinant plasmid is then purified for further subcloning or characterization.
 
Flow Chart

The table below describes the major steps necessary to clone your gene of interest into pCR^® 2.1.
 

When using the TA Cloning^® Kit for the first time, we recommend that you perform the control reactions to help you evaluate your results.

Guidelines for PCR

Generally 10-100 ng of DNA is sufficient to use as a template for PCR. If amplifying a pool of cDNA, the amount needed will depend on the relative abundance of the message of interest in your mRNA population. For optimal ligation efficiencies, we recommend using no more than 30 cycles of amplification.
 
Materials Supplied by the User

• DNA template and primers for PCR product
• Taq polymerase and appropriate 10X PCR buffer 
• Thermocycler

You will need the following reagents and equipment.
 
Polymerase Mixtures

If you wish to use a mixture containing Taq polymerase and a proofreading polymerase, Taq must be in excess of a 10:1 ratio to ensure the presence of 3’ A-overhangs on the PCR product. We recommend using our Platinum^® Taq DNA Polymerase High Fidelity.

If you use polymerase mixtures that do not have enough Taq polymerase or a proofreading polymerase only, you can add 3’ A-overhangs using the method in the Appendix.
 
Producing PCR Products

Perform the PCR in a 50 µl volume containing:

Gel Purification

If you do not obtain a single, discrete band from your PCR, you may gel-purify your fragment before proceeding. 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). Our PCR Optimizer™ Kit (Catalog no. K1220-01) can help you optimize your PCR. Contact Technical Service for more information.

1. Centrifuge one vial of pCR^® 2.1 to collect all the liquid in the bottom of the vial.


2. Determine the volume of PCR sample needed to reach the required amount of PCR product (see above). Use sterile water to dilute your PCR sample if necessary.


3. Set up the 10 µl ligation reaction as follows:

Fresh PCR product                                                                            X µl
10X Ligation Buffer                                                                            1 µl
pCR ^® 2.1 vector (25 ng/µl)                                                                 2 µl
Water                                                                  to a total volume of 9 µl
T4 DNA Ligase (4.0 Weiss units)                                                      1 µl
Final volume                                                                                     10 µl
                                                                                
 
4. Incubate the ligation reaction at 14°C for a minimum of 4 hours (preferably overnight). Proceed to Transforming Competent Cells.

• Chemically competent E. coli suitable for transformation
• S.O.C. medium (warmed to room temperature)
• Positive control, optional (e.g. pUC19)
• LB plates containing 50 µg/ml kanamycin or 100 µg/ml ampicillin (two for each transformation)
• 42°C water bath
• 37°C shaking and non-shaking incubator

• Equilibrate a water bath to 42°C.
• Bring the S.O.C. medium to room temperature.
• If you are using INVaF´ or TOP10 cells, take LB plates containing antibiotic and equilibrate at 37°C for 30 minutes.  Spread each plate with 40 µl of 40 mg/ml X-Gal. Let the liquid soak into the plates.
• If you are using TOP10F´ cells, take LB plates containing antibiotic and equilibrate at 37°C for 30 minutes. Spread 40 µl each of 100 mM IPTG and 40 mg/ml X-Gal onto the plates. Let the liquid soak into the plates.

1. Centrifuge vials containing the ligation reactions briefly and place them on ice.


2. Thaw on ice one 50 µl vial of frozen One Shot^® Competent Cells for each transformation.


3. Pipette 2 µl of each ligation reaction directly into the vial of competent cells and mix by stirring gently with the pipette tip.


4. Incubate the vials on ice for 30 minutes. Store the remaining ligation mixtures at -20°C.


5. Heat shock the cells for 30 seconds at 42°C without shaking. Immediately transfer the vials to ice.


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


7. Shake the vials horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator.


8. Spread 10 µl to 200 µl from each transformation vial on LB agar plates containing X-Gal and 50 µg/ml of kanamycin or 100 µg/ml ampicillin. Be sure to also include IPTG if you are using TOP10F´ cells. We recommend plating 10-50 µl for TOP10F´ or TOP10 cells and 50-200 µl for INVaF´ cells. Note: 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 S.O.C. to allow even spreading.

Analyzing Positive Clones

1. Pick at least 10 white colonies for plasmid isolation and restriction analysis.


2. Grow colonies overnight in 2-5 ml LB broth containing either 100 µg/ml of ampicillin or 50 µg/ml kanamycin.


3. Plasmid and analyze by restriction mapping or sequencing for orientation of the insert. We recommend using the PureLink™ HQ Mini Plasmid Purification Kit for purifying your plasmid DNA.

 
Sequencing Your Insert

If you wish to sequence your insert in pCR^® 2.1, you may use the M13 Reverse Primer to sequence into your insert from the lac promoter. To sequence into the insert from the lacZ_a fragment, you can use either the T7 Promoter Primer or the M13 Forward Primer. Refer to the map of pCR^® 2.1 for the primer sequences and location of the primer binding sites. For your convenience, we offer a custom primer synthesis service.
 
If you have problems obtaining transformants or the correct insert, perform the control reactions as described. These reaction swill help you troubleshoot your experiment. Refer to the Troubleshooting section for additional tips.
 
Long-Term Storage

Once you have identified the correct clone, be sure purify the colony and make 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 100 µg/ml ampicillin or 50 µg/ml kanamycin.

  2.   Isolate a single colony and inoculate into 1-2 ml of LB containing 100 µg/ml ampicillin or 50 µg/ml 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. 

If you do not obtain the results you expect, use the following table to troubleshoot your experiment. We recommend performing the control reactions to help you evaluate your results.

Explanation of Control Reactions

The following table describes the control reactions that can be performed to troubleshoot your TA Cloning^® experiment and how to interpret the results from these control reactions.

Performing the Self-Ligation Reaction

The TA Cloning^® vector is stable for six months if not subjected to repeated freeze-thaw cycles. Vector that has been stored for longer periods or repeatedly frozen and thawed will lose the 3´ T-overhangs resulting in "false" white positives. Follow the protocol below to perform the self-ligation reaction and transform One Shot^® Competent Cells. If you are using another E. coli strain, follow the manufacturer’s instructions.
 
Procedure

1. Set up the 10 µl self-ligation reaction as follows:



^®




2. Incubate overnight at 14-15°C. Centrifuge the vials containing the ligation reactions briefly and place them on ice.


3. Thaw on ice one 50 µl vial of frozen One Shot^® Competent Cells for each transformation.


4. Pipette 1 µl of the Control Ligation Reaction from Step 1, above, directly into the vial of competent cells and mix by stirring gently with the pipette tip.


5. Incubate the vial on ice for 30 minutes. Store the remainder of the ligation mixture at -20°C.


6. Heat shock cells for 30 seconds at 42°C without shaking. Immediately transfer vials to ice.


7. Add 250 µl of room temperature S.O.C. medium to the vial.


8. Shake the vial horizontally at 37°C for 1 hour at 225 rpm in a shaking incubator.


9. Spread 50 µl from the vial on a labeled LB agar plate containing 50 µg/ml of kanamycin or 100µg/ml ampicillin and X-Gal. Be sure to include IPTG if you are using TOP10F´.


10. Incubate plates overnight at 37ºC.
    
     
    Expected Results
    
    You should expect about 5-25 blue colonies from the 50 µl plated. There should be less than 5% white colonies which result from supercoiled pCR^® 2.1 vector. Over time, the 3´ T-overhangs will degrade, causing a blunt-end self-ligation of the vector. This can cause a frameshift of the lacZ gene, resulting in a "false" white or light blue colony with no insert.
     
    Performing the Control Reactions
    
    We recommend performing the control reactions the first time you use the kit to help you evaluate your results. Performing the control reactions involve producing a control PCR product using the reagents included in the kit and using this product in a ligation reaction.
     
    Producing the Control PCR Product
    
    TOP
    
    Use Taq Polymerase and the protocol below to amplify the control PCR product.
    
      1.   Set up the 50 µl PCR as follows:
    
    Control DNA Template (100 ng)                        1 µl
    10X PCR Buffer                                                5 µl
    50 mM dNTPs                                                 0.5 µl
    Control PCR Primers                                          1 µl
    Water                                                           41.5 µl
    Taq Polymerase (1 unit/µl)                                1 µl
    Total Volume                                                   50 µl
    
      2.   Overlay with 70 µl of mineral oil.
    
      3.   Amplify using the cycling parameters below:
    
    

    Step	Time	Temperature	Cycles
    Denaturation	1 minute	94°C	25X
    Annealing	1 minute	55°C	25X
    Extension	1 minute	72°C	25X
    Final Extension	7 minutes	72°C	1X

    
      4.   Remove 10 µl from the reaction and analyze by agarose gel electrophoresis. A discrete 700 bp band should be visible.
    
    Control Ligation Reaction
    
    Using the control PCR product produced, set up the following ligation reaction. In general, 1 µl of the Control PCR Product should be sufficient for ligation. Alternatively, you may use the formula given in Cloning into pCR^® 2.1 to estimate the amount of PCR product to ligate with 50 ng of pCR^® 2.1.
    
    
    1. Set up the 10 µl Control Ligation Reaction as follows:
    
    
    
    ^®
    
    
    
    
    
    2. Incubate the Control Ligation Reaction at 14°C for a minimum of 4 hours (preferably overnight).
    
    
    3. Transform 1 µl of the Control Ligation Reaction into one vial of One Shot^® Competent Cells or into another suitable competent E. coli strain.
    
    
    4. Plate 10-50 µl of each transformation mix on LB agar plates containing 50 µg/ml kanamycin with X-Gal (and IPTG for TOP10F´ cells).
    
    
    5. Incubate plates overnight at 37°C.
        
       Transformation Control
       
       TA Cloning^® Kits supplied with One Shot^® Competent Cells will also be supplied with pUC19 plasmid for use as a transformation control. Transform one vial of One Shot^® cells with 10 pg of pUC19 using the protocol in Transforming Competent Cells. Plate 10 µl to 50 µl of the transformation mixture on LB plates containing 100 µg/ml ampicillin. Transformation efficiency should be 1 x 10⁹ cfu/µg DNA for TOP10F´ and TOP10 cells and 1 x 10⁸cfu/µg DNA for INVaF´.
        
       Expected Results
       
       The Control Ligation Reaction should produce >80% white colonies. Over time, the 3´ T-overhangs will degrade, causing an increase in the number of background white colonies (those without inserts). The number of background colonies should not exceed 10% (see Performing the Self-Ligation Reaction). If this occurs, use another vial of pCR^® 2.1 and avoid repeated freeze-thaw cycles.
        
        
       Adding 3´ A-Overhangs
       
       Direct cloning of DNA amplified by proofreading polymerases into pCR^® 2.1 is often difficult due to very low cloning efficiencies. These low efficiencies are caused by the 3´ to 5´ exonuclease proofreading activity that removes the 3´ A-overhangs necessary for TA Cloning^®. We have developed a simple method to clone these blunt-ended fragments.
       If you routinely clone blunt PCR products, we recommend the Zero Blunt^® PCR Cloning Kit (Catalog nos. K2700-20 and K2750-20) for optimal cloning of blunt PCR products.
        
       Materials Supplied by the User
       
       You will need the reagents and equipment.
       
       
       • Taq polymerase
       • A heat block equilibrated to 72°C
       • Phenol-chloroform
       • 3 M sodium acetate
       • 100% ethanol
       • 80% ethanol
       • TE buffer
        
       Procedure
       
        
       1. After amplification with a proofreading 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. Extract immediately with an equal volume of phenol-chloroform.
       
       
       4. Add 1/10 volume of 3 M sodium acetate and 2X volume of 100% ethanol.
       
       
       5. Centrifuge at maximum speed for 5 minutes at room temperature to precipitate the DNA.
       
       
       6. Remove the ethanol, rinse the pellet with 80% ethanol, and allow to air dry. 
          TOP

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