Cloning Polymerase Chain Reaction (PCR) Products: TA Cloning
Abstract
The nontemplate-dependent terminal transferase activity inherent in nonproofreading DNA polymerases such as Taq provides a highly efficient method to clone PCR products. The enzyme adds a single, unpaired residue—preferentially an adenosyl residue—to each 3′ end of a double-stranded amplified product. The unpaired terminal (A) residues can pair with a linear T vector that carries an unpaired 3′-thymidyl residue at each end. The two chief advantages of TA cloning are speed and lack of reliance on restriction enzymes. The major disadvantage is an inability to clone directionally. For this reason, it is important to pick and analyze several transformed clones when a particular orientation of the amplified fragment is required.
MATERIALS
Reagents
Agarose gels (and other reagents and equipment as required) (see Step 5)
ATP (10 mm)
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Omit ATP from the ligation reaction in Step 1 if the ligation buffer contains ATP.
Bacteriophage T4 DNA ligase and buffer
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See Introduction: Ligation and Ligases (Green and Sambrook 2019a). Some commercial ligase buffers contain ATP; when using such buffers, addition of ATP is not required.
SOB, SOC, or LB agar plates containing the appropriate antibiotic
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If blue/white screening is to be used, the plates should also contain X-Gal and IPTG (see Protocol: Screening Bacterial Colonies Using X-Gal and IPTG: α-Complementation [Green and Sambrook 2019b]).
Target DNA (25 ng/mL), amplified by PCR catalyzed by a nonproofreading thermostable DNA polymerase (e.g., Taq)
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Typically, only ∼30% of the amplified products will carry an unpaired 3′-adenosyl residue. To maximize the efficiency of Taq-catalyzed addition of an unpaired A residue use primers carrying either a 5′-G residue or a 5′-A residue (Magnuson et al. 1996). The efficiency of the nontemplated addition reaction can be improved by programming a 10-min incubation at 72°C at the end of the PCR amplification cycles.
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Before cloning, check the size of an aliquot of the PCR product by gel electrophoresis. When the PCR generates more than one or two bands of amplified DNA, purify the target fragment by electrophoresis through low-melting/gelling-temperature agarose. If not purified by gel electrophoresis, PCR-amplified DNA may be prepared for ligation by extracting the PCR with phenol:chloroform and precipitating the amplified DNA with ethanol. However, many researchers will prefer to use a commercial cleanup kit, such as Wizard SV Gel and PCR Clean-Up System (Promega), PureLink PCR purification kit (Life Technologies), Milllipore Ultrafree spin columns, or QIAquik PCR purification kit (QIAGEN). Some researchers recommend using the PCR products within a day or two of synthesis in insurance against loss of the protruding A-residue. Both T vectors and target DNAs tend to lose their unpaired 3′-residues during prolonged storage at 4°C and when frozen and thawed many times.
T vector
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For synthesis of T vectors, see Protocol: Cloning Polymerase Chain Reaction (PCR) Products: Making T Vectors (Green and Sambrook 2021).
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Ready-made T-vectors can be purchased from many commercial suppliers as components of cloning kits (e.g., pCR-Script [SK+] from Stratagene; pCRII in the TA Cloning kit from Life Technologies; pGEM-T from Promega) (Hengen 1995).
Transformation-competent E. coli cells (and other reagents and equipment for transforming E. coli as required, described in Protocol: The Hanahan Method for Preparation and Transformation of Competent Escherichia coli: High-Efficiency Transformation [Green and Sambrook 2018] and Protocol: The Inoue Method for Preparation and Transformation of Competent Escherichia coli: “Ultracompetent” Cells [Green and Sambrook 2020a])
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Transformation-competent E. coli cells can be bought commercially or prepared as described in Protocol: The Hanahan Method for Preparation and Transformation of Competent Escherichia coli: High-Efficiency Transformation (Green and Sambrook 2018), Protocol: The Inoue Method for Preparation and Transformation of Competent Escherichia coli: “Ultracompetent” Cells (Green and Sambrook 2020a), Protocol: Easy Transformation of Escherichia coli: Nanoparticle-Mediated Transformation (Green and Sambrook 2019c), and Protocol: Transformation of Escherichia coli by Electroporation (Green and Sambrook 2020b).
Equipment
Incubator (37°C)
Water bath (preset to 14°C)
METHOD
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For more information on this method, see Clark (1988); Holton and Graham (1991); Marchuk et al. (1991); Trower and Elgar (1994); Zhou and Gomez-Sanchez (2000).
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1. In a microcentrifuge tube, set up a ligation reaction.
Amplified target DNA (25 ng/µL) 1 µL T-tailed plasmid (75 ng/µL) 1 µL Ligation buffer (10×) 1 µL Bacteriophage T4 DNA ligase 3 U H2O to 10 µL -
The optimal molar ratio of vector:amplified DNA in the ligation mixture is 3:1. The amounts of the two DNAs suggested for the ligation reaction (above) assume that the vector and amplified DNA are of equal size. This will rarely be the case. When the size of the target DNA is known, the relative amounts of the two types of DNA included in the ligation reaction should be altered to achieve a molar ratio of 3:1 (vector:amplified DNA).
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If necessary, add ATP to a final concentration of 1 mm. Set up a control reaction that contains all the reagents listed above except the amplified target DNA.
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2. Incubate the ligation mixtures for 4 h at 14°C.
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3. Dilute 5 µL of each of the two ligation mixtures with 10 µL of H2O and transform a suitable strain of competent E. coli to antibiotic resistance as described in Protocol: The Hanahan Method for Preparation and Transformation of Competent Escherichia coli: High-Efficiency Transformation (Green and Sambrook 2018), Protocol: The Inoue Method for Preparation and Transformation of Competent Escherichia coli: “Ultracompetent” Cells (Green and Sambrook 2020a), Protocol: Easy Transformation of Escherichia coli: Nanoparticle-Mediated Transformation (Green and Sambrook 2019c), and Protocol: Transformation of Escherichia coli by Electroporation (Green and Sambrook 2020b). Plate the transformed cultures on media containing the appropriate antibiotic and, depending on the genotype of the host and vector, IPTG and X-Gal (see Protocol: Screening Bacterial Colonies Using X-Gal and IPTG: α-Complementation [Green and Sambrook 2019b]).
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4. Calculate the number of colonies obtained from each of the ligation mixtures. If the plasmid is equipped for blue/white screening, pick a number of white colonies obtained by transformation with the ligation reaction containing the target DNA. In different experiments, the ratio of blue:white colonies can vary between 1:5 and 2:1.
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5. Confirm the presence of the amplified fragment by isolating the recombinant plasmid DNAs and digesting them with the appropriate restriction enzymes. Fractionate the restricted DNA by electrophoresis through an agarose gel using appropriate DNA size markers.
Footnotes
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From the Molecular Cloning collection, edited by Michael R. Green and Joseph Sambrook.
