Gotaq polymerase kit

Selected ZMYND fragments were PCR-amplified from the genomic DNA or cDNA. PCR primers, designed with the use of Primer3 software, flanked whole exons or those exon fragments where the genotyped mutations were localized; to check if there were no SNPs in the primer binding sites, an SNPcheck v3 was used. The amplicon size for HRM analysis and sequencing, respectively, was 90–150 bp or 250–300 bp. A standard PCR amplification preceding dideoxy sequencing or cDNA analysis was carried out using: 0.4 ng/μl of DNA or cDNA template, 0.4 pM of each of the primers (forward and reverse), 0.12 mM deoxynucleoside triphosphates (Invitrogen) and a GoTaq polymerase kit (Promega); PCR reactions were conducted for 32 cycles with annealing temperatures of 60°C or 61°C. Real-time PCR reactions used in HRM analysis are described in one of the following sections. All primer pairs are listed in S1 Table.

Total RNA was prepared with the NucleoSpin RNA kit (Macherey -Nagel) and treated with DNase (Thermo Scientific). RNA (1 μg) was reverse transcribed into cDNA via Oligo (dT) with SuperScript III Reverse Transcriptase (Invitrogen). PCR was perfomed using Go Taq polymerase kit (Promega) with following conditions; 95 °C for 2 min; followed by 34 cycles of 94 °C for 30 s, 60 °C for 30 s, and 72 °C for 45 s; followed by a single cycle of 72 °C for 5 min using the primers [37 (link)] given below.

Primers	Band size (bp)	Tm (0C)
Oct4-F: aac ctg gag ttt gtg cca ggg ttt	120	60
Oct4-R: tga act tca cct tcc ctc caa cca
Tbrachyury-F: tgt ccc agg tgg ctt aca gat gaa	140	60
Tbrachyury-R: ggt gtg cca aag ttg cca ata cac
ISL1-F: cac aag cgt ctc ggg att gtg ttt	200	60
ISL1-R: agt ggc aag tct tcc gac aa
Nkx2.5-F: gcg att atg cag cgt gca atg agt	220	60
Nkx2.5-R: aac ata aat acg ggt ggg tgc gtg
cTNT-F: ttc acc aaa gat ctg ctc ctc gct	160	60
cTNT-R: tta tta ctg gtg tgg agt ggg tgt gg
β-actin (BA)-F: ttt gaa tga tga gcc ttc gtc ccc	130	60
β-actin (BA)-R: ggt ctc aag tca gtg tac agg taa gc

Total RNA was isolated from HeLa-MAGI-CCR5 cells transfected with indicated amounts of the Tat/pcDNA 3, Flag PACT/pcMV2, Flag TRBP/pcDNA 3.1⁻ or pcDNA 3.1 expression constructs. After two washes with ice-cold PBS, 250 µl of RNAZol B was added and total RNA was isolated as per the manufacturer's instructions. cDNA was synthesized at 42°C for 1 h using random hexamer primers, 1 µg of total RNA, M-MuLV reverse transcriptase, 500 µM dNTPs and RNase inhibitor RNAsin (Promega). For each PCR, 2 µl of cDNA and 50 pmoles of forward and reverse primers designed to amplify a 166 bp region of the β-galactosidase transcript or a 500 bp region of the β-actin transcript were used with the Promega GoTaq Polymerase Kit. The following conditions were used for PCR: 95°C for 5 min (initial denaturation), denaturation at 95°C for 30 s, annealing at 45°C for 30 s and extension at 72°C for 30 s for 20, 25 or 30 cycles.