The CCDC170 3' UTR containing the rs9383935 G allele was amplified by PCR from human genomic DNA carrying the GG homozygous genotype template with the following primers: sense 5'-AGACGCGTTAAGTCAGGGGCTTTACTAGC-3' and antisense 5'-GCAAGCTTCTGCTGAGTAGTTGGGATTACA-3'. The PCR products were separated in agarose gel, extracted, purified and cloned into the pMIR-REPORT™ miRNA expression reporter vector system (Applied Biosystems, Foster City, CA, USA) with MluI and HindIII digestion and then were ligated by T4 DNA ligase to the recombinant constructs (Additional file 2 : Figure S2). The plasmid with the rs9383935 G allele was used as the template for the mutation G → A. The site-directed mutagenesis for the plasmid with the A allele construct was generated using a Mut Express Fast Mutagenesis kit (Vazyme Biotech, Nanjing, China). All PCR amplifications and constructs were sequenced to confirm the accuracy of cloning.
Mut express fast mutagenesis kit
Manufactured by Vazyme
Sourced in China
The Mut Express Fast Mutagenesis kit is a laboratory tool designed for efficient site-directed mutagenesis. It provides a convenient and reliable method for introducing specific mutations into DNA sequences.
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Lab products found in correlation
2 protocols using mut express fast mutagenesis kit
1
Cloning the CCDC170 3' UTR with rs9383935
2
Identifying Isoeugenol and Eugenol Determinants
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To identify the specific residues determining whether the product is isoeugenol or eugenol, we conducted sequence comparisons of ScIGS1, PhIGS1, CbIGS1, PhEGS1, CbEGS1, CbEGS2, and ObEGS1 using CLC Genomics Workbench. The mutagenic primers (Supplementary Table 2 ) for each mutation were designed using an online tool (https://crm.vazyme.com/cetool/singlepoint.html ). Subsequently, the ScIGS1 mutants were constructed in the pET-28a expression vector using the Mut Express Fast Mutagenesis Kit (Vazyme, Nanjing, China). The mutations were then transformed into E. coli DH5α competent cells and confirmed by sequencing in Tsingke Biotechnology. All ScIGS1 mutant proteins were individually heterologously expressed and assayed as described before, except that a multistep gradient from 95 % A (0–3 min), 95-70 % A (3–7 min), 70-25 % A (7–17 min), 25-5% A (17–27 min), and 5 % A (27–32 min). In addition, the enzyme turnover numbers (kcat) of ScIGS1 and its mutants were predicted using a deep learning approach based merely on substrate structure and protein sequences [49 (link)].
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