Iii reverse transcriptase

To determine the 5′-terminal sequence of traA mRNA, cDNA was synthesized using total RNA of Anc(C) as the template, SuperScript®; III reverse transcriptase (Life Technologies, Carlsbad, CA, USA), and traA_r primer. The 5′-phosphorylated DNA linker 5PpACYC_rev was ligated at the 3′-terminus of the first strand cDNA with T4 RNA ligase 1 (New England Biolabs). PCR was performed using the resultant cDNA as the template, PrimeSTAR®; HS DNA polymerase (Takara Bio Inc., Shiga, Japan), and the primers pACYC_rev2 and traA_r2. To determine the 3′-terminal sequence of traA mRNA, universal miRNA cloning linker (5′-rAppCTGTAGGCACCATCAAT-NH2-3′; New England Biolabs) was ligated with the 3′-terminus of total RNA of Anc(C) using T4 RNA ligase 1 (New England Biolabs). The first strand cDNA was synthesized using the primer Linker_r and SuperScript®; III reverse transcriptase (Life Technologies), and then purified cDNA was subjected to PCR using the primers Linker_r and traA_f. Three and two bands of PCR products for 5′- and 3′-terminal sequence determination, respectively, were sliced from the gel and subcloned using a Zero Blunt®; TOPO®; PCR Cloning Kit for Sequencing (Life Technologies). Three to six clones were randomly picked and sequenced by the dideoxynucleotide chain termination sequencing method.