Thermalace dna polymerase

Manufactured by Thermo Fisher Scientific

ThermalAce DNA Polymerase is a highly thermostable DNA polymerase enzyme suitable for use in PCR amplification. It exhibits robust performance and enhanced fidelity compared to Taq DNA polymerase.

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Amplitaq gold 360 polymerase, by Thermo Fisher Scientific (1 mentions)

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DNA polymerase (71 citations)

2 protocols using thermalace dna polymerase

Plasmid Construction for Repeat Expansions

Cited 2 times

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The (G₄C₂)₆₆, (G₄C₂)₆₆-NoATG-GFP and (G₄C₂)₆₆-NoATG-Nanoluc plasmids were constructed using previously described methods (Gendron et al., 2013 (link); Yang et al., 2015 (link)). To generate the (TG₃C₂)₆₂ plamsid, gDNA from fibroblasts of a Sca36+ patients was used as a templates in a nested PCR strategy using ThermalAce DNA Polymerase (Invitrogen) or AmpliTaq Gold 360 Polymerase (Thermo Fisher). The sequence includes 69 bp 5’ of the repeat expansion and 40 bp of 3’ flanking sequence. The PCR products were cloned into the pAG3 expression vector (gift of T. Golde, UF), then sequentially ligated using TypellS restriction enzymes to generate a (TG₃C₂)₆₂ fragment. All TGGGCCn fragments with 5’ and 3’ flanking sequences were subcloned into the pAG₃ expression vector containing two upstream stop codons in each reading frame, as well as 3 different C-terminal tags in alternate frames [i.e., (GP)n-HA, (GL)n-Myc and (WA)n-FLAG].

Wang Z.F., Ursu A., Childs-Disney J.L., Guertler R., Yang W.Y., Bernat V., Rzuczek S.G., Fuerst R., Zhang Y.J., Gendron T.F., Yildirim I., Dwyer B.G., Rice J.E., Petrucelli L, & Disney M.D. (2018). The Hairpin Form of r(G4C2)exp in c9ALS/FTD is Repeat-associated non-ATG Translated and a Target for Bioactive Small Molecules. Cell chemical biology, 26(2), 179-190.e12.

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Generating G4C2 Repeat Expression Vectors

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To generate the G₄C₂ repeat 423 and 774 expression vectors, we used muscle or spleen DNA from an affected C9orf72 expansion carrier as a template in a nested PCR strategy. We used ThermalAce DNA Polymerase (Invitrogen) to amplify the 66 G₄C₂ repeat region of a previously constructed G₄C₂ repeat plasmid, including 113 and 99 nucleotides of 5′ and 3′ flanking sequence, respectively. We then used these intermediate plasmids to construct PCR products for the 423 and 774 sequence that were subsequently cloned into the pAG3 and pcDNA6 expression vectors containing 3 different C-terminal tags in alternate frames. The EGFP gene and SCA36 ‘GGCCTG’ 66 repeat were cloned into pAAV expression vectors. The SCA36 clone was Sanger sequenced to determine the number of repeats, but the others were too long for Sanger sequencing.

Ebbert M.T., Farrugia S.L., Sens J.P., Jansen-West K., Gendron T.F., Prudencio M., McLaughlin I.J., Bowman B., Seetin M., DeJesus-Hernandez M., Jackson J., Brown P.H., Dickson D.W., van Blitterswijk M., Rademakers R., Petrucelli L, & Fryer J.D. (2018). Long-read sequencing across the C9orf72 ‘GGGGCC’ repeat expansion: implications for clinical use and genetic discovery efforts in human disease. Molecular Neurodegeneration, 13, 46.

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