Dna engine tetrad 2 peltier thermal cycler

All positive samples were cleaned and sequenced by a commercial company (Macrogen Spain, Madrid) using DNA Engine Tetrad 2 Peltier Thermal Cycler (BIO-RAD) and ABI BigDye^® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA), following the manufacturer’s protocol. The obtained sequences were manually checked and aligned with Molecular Evolutionary Genetics Analysis (MEGA X) software and FinchTV 1.4.0 software (Geospiza, Inc.; Seattle, WA, USA). The consensus sequences of each positive sample were then compared with other available sequences using the BLAST tool (National Library of Medicine, Rockville, MD, USA) [21 ]. All obtained sequences of more than 200 base pairs were submitted to GenBank for accession number identification.

For all seven IL7R network genes the suggested Taqman® assay (Applied Biosystems, Bleiswijk, the Netherlands) was selected. Reverse transcription was performed with total RNA from blood of the 745 samples that passed QC using the First Strand cDNA Synthesis Kit, according to the manufacturer’s protocol (Roche Applied Science, Almere, the Netherlands). cDNA was amplified using the DNA Engine Tetrad® 2 Peltier Thermal Cycler (Bio-Rad, Veenendaal, The Netherlands). qPCR was then performed with the Taqman® method using the BiomarkTM 48.48 and 96.96 Dynamic Arrays (Fluidigm Amsterdam, The Netherlands). Relative gene expression of the BioMark™ Array data were calculated by using the 2^−ΔΔCt method, in which Ct indicates cycle threshold, the fractional cycle number where the fluorescent signal reaches the detection threshold [42 (link)]. YKT6 was used as internal control and commercially available human total reference RNA (Clontech Laboratories, Mountain View, CA, USA) as reference RNA.

Isolates were grown in Tryptic Soy agar (TSA) plates (Difco, BD, Sparks, MD). A single colony was grown in a shaking incubator overnight at 37°C in Brain Heart Infusion broth (BHIB) (Difco). One ml of the broth culture was transferred to a 1.5 ml microcentrifuge tube and centrifuged at 12,000 rpm for 3 min. Total genomic DNA was isolated using the Promega Wizard Genomic DNA Purification kit (Promega Corporation, Madison, WI). The primers used for the amplification of the selected genes are described in Table S1. DNA amplification by PCR was performed in a reaction volume of 50 μl consisting of 25 μl of Qiagen Hot StarTaq Plus master mix (Qiagen, Valencia, CA), 20 μM primer mix, 10 ng of total genomic DNA and volume was completed with molecular biology grade water. Initial denaturation was carried out for 5 min at 95°C. Thirty cycles of amplification were performed in a DNA Engine Tetrad2 Peltier Thermal Cycler (Bio-Rad, Hercules, CA). Each cycle consisted of three steps: denaturation for 30 sec at 94°C, annealing for 30 s at 60°C, and extension for 1 min at 72°C. An additional step of extension for 7 min at 72°C was performed at the end of the amplification to complete extension of the primers. Amplification products were detected by resolving 1 μl of the PCR product using the Agilent DNA 7500 kit and the 2100 Agilent Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA).

Total RNA was harvested from PANC-1 cells using a NucleoSpin RNA isolation kit (Macherey-Nagel) on a freedom evo robot (Hamilton). cDNA was synthesized on a DNA Engine Tetrad 2 Peltier Thermal Cycler (BioRad) on a Hamilton LiHa robot.
Quantitative real-time PCR was performed on a ViiA7 Real-time PCR System (Applied Biosystems) according to the manufacturer's protocol. The reactions were carried out in 10 μL reaction volume on 384 well plates. Samples were analyzed in triplicates. RT-PCR results were analyzed using the 2^-ΔΔCt method [22 (link)], the expression of HMBS (hydroxymethyl-bilane synthase) and to the experimental control afterwards. Primers and probe mixtures were acquired from Applied Biosystems.

Genomic DNAs were extracted from young rice seedlings using a modified CTAB DNA extraction procedure (Murray and Thompson 1980 (link)). PCR was performed in a reaction volume of 20 μL containing 40 ng of genomic DNA, 0.2 μM of each primer, 200 μM of each dNTP, 10 mM Tris–Cl (pH 8.3), 50 mM KCl, 1.5 mM MgCl₂, 0.01 % gelatin, and 0.5 U of Taq DNA polymerase. PCR amplifications were carried in a DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad) using the following program: 5 min at 94 °C; 32 cycles of 45 s at 94 °C, 30 s at 47–65 °C, 20 s at 72 °C; and 10 min final extension at 72 °C (Additional file 7). Amplified PCR products were separated on a 1–3 % agarose gel to validate the expected fragment size.

First, DNA from cells and tissues was extracted using the QIAmp DNA Mini kit (Qiagen, German), according to the manufacturer’s recommendations. Next, we used the EpiTect Bisulfite kit (Qiagen) for bisulfate conversion following the product manual. At last, Methylation specific PCR (MSP) was performed on Bisulfite-treated DNA using EpiTect MSP kit (Qiagen). The primers of MEG3 promoter which specific for methylated and unmethylated were as follows: the methylated pair (M) was 5′-GTT AGT AAT CGG GTT TGT CGG C (forward) and 5′-AAT CAT AAC TCC GAA CAC CCG CG (reverse); the unmethylated pair (U) was 5′-GAG GAT GGT TAG TTA TTG GGG T (forward) and 5′-CCA CCA TAA CCA ACA CCC TAT AAT CAC A (reverse). The PCR reaction was conducted in DNA Engine Tetrad 2 Peltier Thermal Cycler (Bio-Rad) under the following conditions: 95 °C 15 min; 94 °C 30s, 70 °C 30s, 72 °C 30s, 5 cycles; 94 °C 30s, 65 °C 30s, 72 °C 30s, 5 cycles; 94 °C 30s, 60 °C 30s, 72 °C 30s, 30 cycles; 72 °C 7 min. The PCR products were identified by electrophoresis through 2.5% agarose gel, stained with ethidium bromide, visualized under UV and compared by densitometry.