Onetaq hot start dna polymerase

Total RNA was isolated from KYSE150 and EC7 cells using the RNeasy Mini Kit (Qiagen, 74104, Wroclaw, Poland) according to the manufacturer’s instructions. cDNA was synthesized from 2 μg of total RNA using the SuperScript IV VILO Master Mix with ezDNase Enzyme (Invitrogen, 11766050). PCR was performed with OneTaq^® Hot Start DNA Polymerase (New England BioLabs, M0481). Cycling conditions were as follows: initial denaturation at 94 °C for 30 s, 30 cycles of 30 s at 94 °C and 30 s at 60 °C, and final extension at 68 °C for 5 min. Each sample was run in triplicate. Amplification efficiency was assessed for all primer sets used in separate reactions, and primers with efficiencies in a range of 95–100% were used. Primers for the LDHA and LDHB genes were products of OriGene (HP208683 and HP208217). In order to normalize gene expression, primers for GAPDH or β-actin (ACTB) (OriGene, HP100003 and HP204660, respectively) were used. The ΔCt was calculated by subtracting the cycle threshold (Ct) value of GAPDH mRNA from the Ct value of the gene of interest (ΔCt). Fold change (FC) in the gene expression was calculated using the 2^−ΔΔCt method.

Total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. One μg RNA was reverse transcribed into cDNA using 500 μM of dNTP (R0193), 5 μM Oligo(dT)18 primer (S0132), 5 μM Random Hexan primer (S0142), 1 U RiboLockTM RNase Inhibitor (E00381) and 5 U RevertAidTM M-MuLV Reverse Transcriptase (EP0441) in the supplied reaction buffer (all reagents from Thermo Scientific, Schwerte, Germany). The cDNA reactions were performed for 10 min/25 °C, 1 h/37 °C and stopped at 72 °C for 10 min. cDNA (2.5 μl) was used as a template with following specific primers (customized by Eurofins, MWG GmbH, Ebersberg, Germany) as described previously [27 (link), 28 , 31 (link)].
PCR reactions included 0.2 μM of each primer, 200 μM of dNTP (R0193, Thermo Scientific) and 0.03 U One Taq Hot Start DNA polymerase (New England Biolabs GmbH, Frankfurt am Main, Germany) in the supplied reaction buffer. PCR cycling conditions were performed 30s at 94 °C, 1 min at 60 °C and 68 °C for 1 min respectively, including an initial 30s denaturation step at 94 °C and a final 5 min extension step at 68 °C (35 cycles). Aliquots of 25 μl of each RT-PCR product were separated on a 2% agarose gel including the standard GeneRuler 100 bp DNA Ladder (Thermo Scientific) and visualized by GelRedTM (Biotium Inc., Hayward, CA, US) staining.

First strand cDNA was generated using the iScript cDNA synthesis kit (Bio-Rad). Quantitative RT-PCR was performed using OneTaq^® Hot Start DNA polymerase (New England Biolabs) and SYBR Green I (Life Technologies). The primers used for Quantitative RT-PCR are listed in Supplementary Data 10.

PCR of genomic AAVS1 regions was carried out with OneTaq hot start DNA polymerase (NEB) as described by the manufacturer, with an initial denaturation step of 4 min and an annealing temperature of 63°C. Primers are listed in (Figure S1).