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Thermo Scientific InsTAclone PCR Cloning Kit is a TA system for direct one-step cloning of PCR products with 3'-dA overhangs (see Reference 1) generated by Taq DNA polymerase and other thermostable DNA polymerases, which lack proofreading activity. The high quality ready-to-use TA cloning vector pTZ57R/T (see Figure 1 in Supporting Data) was prepared by linearizing the pTZ57R plasmid with Eco32I and tailing with single ddT. The 3'-ddT overhangs at both ends of the cloning site prevent recircularization of the vector during ligation, resulting in high cloning yields and low background.
To increase the speed, convenience, and efficiency of cloning experiment, the kit has been combined with the unique TransformAid Bacterial Transformation Kit – a set of solutions for preparation of chemically competent E. coli cells.
According to the protocol, ligation and preparation of competent cells is performed in parallel. Therefore, it takes only one hour from the completion of PCR to cell plating. Our transformation protocol is often faster than the transformation of commercially available competent cells. The DNA insert can be readily excised from the versatile polylinker of pTZ57R/T, sequenced using standard M13/pUC primers or in vitro transcribed with T7 RNA polymerase.
>Sequence (953bp) of Control PCR Product (from #K1213, #K1214 InsTAclone™ PCR Cloning Kit) aatggatccttaaaaatctcccgaagcgacttgccagcattcaacaccgatacgacgccacatttcaact acttgagttcggtcatcaatagctaatttcacgtcaaaatgcggtgcaatgtgtttccagaaaatttctt ctttaactacatcgtctttacgggtatcgccttgttcgcgctgacattgcataactaatggaacgccagc aatgtcctcaacccatttacgggtcatacgataatatttcgttgggtcttctttagttccactttcacga cctgaaacgactacgatttgataacccataagagcatacatcttagacagttcaacaaccataggattga taacatcggtatcgcatttttcaaggtcataaggaccacgaccattcattttagctagtgtaccatcaac atcaaaaataactgcttttggtttaccaggagtcccattatatactggaagaccgagatactctcgcatg cttttatacattgaacgtaaaacatcaattggtactgctttagttccgcgttttgagttacgtttaacca attcagtccaaggaacatcaaacactttatgttcaactttccagccgtattctttggcaaaagtttccca tgctaggcgacgttcaggattcaggttagtatctgaaatgattactcccttaacagaatcgccaccgtac agaatacttttagctgtatcaaactgcataccagttacgataccttctttctttttggtatacttgtact catcgcgttcttcatgcgccataatagattggcgatagtcatcacgattgatattataaaacccgggatt cttagcaataaattcacgagcccaagtactcttaccagaaccaggacagccaatagtcaaaataatcttt ttcatttattttttctcaactaatgattgaatatccggatgtt
> pTZ57R Plasmid sequence (2886 bp) gacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccct agcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggc tccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtggg ccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactgg aacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatg agctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttccattcgccattcaggctgcgca actgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgatta agttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgaattcgagctcggtacctcgcgaat gcatctagatatcggatcccgggcccgtcgactgcagaggcctgcatgcaagctttccctatagtgagtcgtattagagc ttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaag cataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagt cgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttcc gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacg gttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaagg ccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcga aacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgct taccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcgg tgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactat cgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggta tgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctc tgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttt tttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgc tcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaatt aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggca cctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggaggg cttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagc cagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagct agagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtt tggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggtta gctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataat tctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtat gcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatca ttggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgca cccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaa gggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttatt gtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtg ccacct
E. coli strains are not included. Suitable for all common laboratory E. coli strains.
Restriction map of vector pTZ57R/T.
PCR product cloning procedure
J. M. Clark, Novel non-templated nucleotide addition reactions catalyzed by prokaryotic and eukaryotic DNA polymerases. Nucl. Acids Res. 16(20), 9677-9686 (1988).