|Phusion Hot Start II DNA Polymerase tends to work better at elevated denaturation and annealing temperatures due to higher salt concentrations in its buffer. Hence follow manufacturer instruction strictly.
Construction of GARS mammalian expression vectors
GARS mRNA sequences were introduced into a modified pcDNA3.1 vector for expression. In order to transcribe mRNAs beginning with their exact 5′-extremity, the “extra” sequence present between the pcDNA3.1 transcription start site and the KpnI restriction site was removed by mutagenesis (Phusion Site-Directed Mutagenesis Kit, Thermo Scientific). The sequence of the GARS ORF was generated by RT-PCR from HeLa cell total RNA, fused to a C-terminal V5 epitope sequence and introduced in the modified pcDNA3.1 between the KpnI and XbaI restriction sites. Then both 5′-UTR sequences were PCR amplified from brain cDNA (Ambion) and introduced between the KpnI restriction site and the unique internal NheI restriction site present at nt 205 in the mitochondrial GARS ORF. Mutants were generated by replacing each putative ATG initiator codon by the ATA sequence (Phusion Site-Directed Mutagenesis Kit). A 5′ stable hairpin structure.19 was introduced at the KpnI site using 2 overlapping primers: 5′-AATTGGTACCTTTGCAAAAAGCTCCACCACGGCCCAAGCTTGGGC-3′ and 5′- TTA-AGGTACCAAGCTCCACCACGGCCCAAGCTTGGG-3′.
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