H taq dna polymerase

Genomic DNAs (gDNA) were extracted from cancer cell lines and were treated with bisulfite using EpiTect Bisulfite Kit (Qiagen) according to the manufacturer’s instruction. Multiplexed PCR was prepared with 113 pairs of primers in total. To design the multiplex PCR primers, promoter sequences between 1 kb upstream and 500 bp downstream from transcription start sites (TSSs) of multiple target genes were extracted using the ‘bedtools getfasta’ (Quinlan, 2002 (link)), and then the C→T and G→A converted target DNA sequences were generated from the extracted sequences by ‘bismark_genome_preparation’ (Krueger and Andrews, 2011 (link)). With these sequences as input templates, we ran a web-based ‘Batchprimer3’ engine (You et al., 2008 (link)) to obtain primers in batches which were then split into six subsets after experimental verifications of their proper operations by gel electrophoreses of multiplex PCR products from various combinations of primer sets. The list of primer pairs and their amplicon sequences is presented in the Supplementary File 1. Multiplex PCR was performed with each primer group using h-Taq DNA polymerase (SolGent) in the following conditions: 15 min of enzyme activation at 95°C followed by 50 cycles of 95°C for 20 s, 46°C for 1 min, and 65°C for 2 min (Oh et al., 2015 (link); Park et al., 2017 (link)).

The midlog phase of bacterial cultures was treated with 1 μmol·ml⁻¹ oxacillin for 24 h and was subsequently used for RNA extraction [28 (link)]. Treatment with RNAse-free DNAse I (Sigma) was performed at 37°C for 2 h. The concentration and DNase-free quality of RNA samples were spectrophotometrically assessed and confirmed by the amplification of chromosomal bla_OXA-51 and 16S rRNA. Fifteen microliters of each RNA sample was reverse-transcribed in a final volume of 20 μl containing random hexamers, MMLV reverse transcriptase (Agilent) at 42^o C for 45 min.
Amplification of bla_OXA-51, bla_OXA-58, and 16S rRNA was performed in a final volume of 25 μl containing 5 μl cDNA, 3 mmol MgCl₂, 200 nmol dNTPs, 2 U h-Taq DNA polymerase (Solgent), 300 nmol of OXA-51/58-F/R primers, 150 nmol of OXA-51/58-P probes, 200 nmol of 16S-F/R primers, and 100 nmol of 16S-P probe (IDT). Primer and probe sequences are given in Table 1. Each real-time PCR was performed in triplicate on the Stratagene Mx3005P real-time PCR system (Agilent). The reaction mixture was incubated for 15 min at 95°C, followed by 40 cycles of 10 s at 95°C and 20 s at 60°C. Normalized expression of bla_OXA-51 and bla_OXA-58 genes was calculated relatively to the 16S rRNA reference gene according to the 2^-ΔΔCt method [29 (link)].

For reverse transcription-polymerase chain reaction (RT-PCR), total RNAs were obtained from cultured cells using RNeasy mini kit (Qiagen) and were used to synthesize cDNAs using reverse transcriptase (Superscript III, Invitrogen) as described elsewhere (Cho et al., 2015 (link)). PCR was performed with h-taq DNA polymerase (SolGent) in the following conditions: 15 min of enzyme activation at 95°C followed by 40 cycles of 95°C for 20 s, 55°C for 30 s, and 65°C for 1 min. The list of PCR primers is as follows: 5′-CAGGGGATGGCAAGATAG-3′ and 5′-TCTTTTATTGTCCACCATCC-3′ for LIFR; 5′-AACAGTGC CTTGAGGAGAG-3′ and 5′-GGGCTGTTTAGGTAATTCG-3′ for LIFR-AS1; 5′-AATCCCATCACCATCTTCCA-3′ and 5′-TG GACTCCACGACGTACTCA-3′ for GAPDH.