Ptc 100 peltier thermal cycler

Genomic DNA was extracted from young and healthy leaf blades of each accession approximately 3 months after germination using the methods described by Gross et al. (2009 (link)). According to the rice molecular map and microsatellite database published by Temnykh et al. (2000 (link)) and McCouch et al. (2002 (link)), 249 SSRs scattered on 12 chromosomes were selected. The SSR primers were synthesized by Shanghai Generay Biotech Co., Ltd. (Shanghai, China).
Each 10-μl PCR reaction contained 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl₂, 0.5 nM dNTPs, 0.14 pM forward primer, 0.14 pM reverse primer, 0.5 U of Taq polymerase, and 20 ng of genomic DNA. DNA amplification was performed using a PTC-100™ Peltier Thermal Cycler (MJ Research™ Incorporated, USA) under the following conditions: (1) denaturation at 94°C for 5 min; (2) 34 cycles of denaturation at 94°C for 0.5 min, annealing at 55–61°C for 1 min, and extension at 72°C for 1 min; and (3) a final extension at 72°C for 10 min. The PCR products were separated on an 8% polyacrylamide gel for 1 h at 150 V and visualized using silver staining. One pair of SSR markers was used to detect one locus, and each polymorphic band at the same marker locus in the population was recorded as one allele. After screening the PAGE products, the size of each band was determined using Quantity One software (Bio-Rad Company, USA).

Genomic DNA was isolated from 300 μL EDTA-treated whole blood using a Commercial kit (Wizard genomic DNA purification kit, Promega, WI, USA). The procedure was carried according to the kit manufacturer's recommendation.
Genotyping analysis for detection of 3 SNPs of IL-10s was performed for all patients by using specific PCR primers. Table 1 describes primers used and PCR conditions. PCR was performed in a total volume of 25 μL using 100 ng of genomic DNA with 1.5 μL of 10 μmol/L of each primer and 12.5 μL of 2X KAPA2G Fast ReadyMix PCR Kit (Kappa Biosystems, USA). PCR amplifications were performed in PTC-100 Peltier Thermal Cycler (MJ Research, MA, USA).
PCR reaction products were sequenced using Big Dye Terminator version 3.1 kit (Applied Biosystems, Waltham, MA, USA). Samples were run on an ABI Prism Genetic Analyzer system 3130xl (Applied Biosystems, Waltham, MA, USA).

Genomic DNA was extracted from leaf tissue of each selected plant according to the methods described by Murray and Thompson (1980 (link)). According to the published rice molecular map and microsatellite database of Temnykh et al. (2000 (link)) and McCouch et al. (2002 (link)), 262 polymorphic microsatellite markers, approximately evenly distributed scattered on 12 chromosomes were used for genotyping (Figure 1). Primers were synthesized by Shanghai Generay Biotech Co. Ltd., Shanghai, China. Each 10 μl PCR reaction consisted of 10 mM tris HCl (PH 9.0), 50 mM KCl, 0.1% triton X-100, 1.5 mM MgCl₂, 0.5 nM dNTPs, 0.14 pM forward primers, 0.14 pM reverse primers, 0.5 U of taq polymerase, and 20 ng of genomic DNA. DNA amplification was performed using a PTC-100™ Peltier Thermal Cycler (MJ Research™ Incorporated, USA) under the following conditions: (1) denaturation at 94°C for 5 min; (2) 34 cycles of denaturation at 94°C for 0.5 min, annealing at 55–61°C for 1 min, and extension at 72°C for 1 min; (3) final extension at 72°C for 10 min. The PCR products were run on 8% polyacrylamide gel at 150 V for 1 h, and visualized using silver staining.

Genomic DNA was extracted from leaf tissue following the methods that are described in Cheng et al. [37 ]. A total of 263 SSR markers that were selected from the rice maps [38 , 39 (link)] were used to genotype 95 rice accessions. PCRs were conducted in a 10-μL reaction mixture containing 1 μL of 20 ng μL^-1 template DNA, 0.6 μL of 25 mmol L^-1 MgCl₂, 0.7 μL of 2 pmol μL^-1 forward primers, 0.7 μL of 2 pmol μL^-1 reverse primers, 0.2 μL of 2.5 mmol L^-1 dNTP, 1 μL of 10×PCR buffer, 0.1 μL of 5 U μL^-1 Taq DNA polymerase (Dongsheng Biotech, China) and 5.7 μL of ddH₂O. DNA amplification was performed using a PTC-100 Peltier Thermal Cycler (MJ Research Inc., USA). The PCR reaction program included denaturation at 95°C for five minutes, followed by 31 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension step at 72°C for five minutes. Electrophoresis and silver staining were performed as described in Liu et al. [40 ].

DNA was extracted from fresh leaves of individuals of the 540 rice accessions, 254 RILs and single segment segregating F₂ population using the method reported by Monna et al. (2002 (link)). A total of 262 SSR markers selected from the rice maps (Temnykh et al., 2000 (link); McCouch et al., 2002 (link)) were used to genotype 540 rice accessions. PCR amplification was conducted in a 10-μL reaction mixture containing 1 μL of 20 ng μL⁻¹ template DNA, 0.6 μL of 25 mmol L⁻¹ MgCl₂, 0.7 μL of 2 pmol μL⁻¹ forward primers, 0.7 μL of 2 pmol μL⁻¹ reverse primers, 0.2 μL of 2.5 mmol L⁻¹ dNTP, 1 μL of 10 × PCR buffer, 0.1 μL of 5 U μL⁻¹ rTaq DNA polymerase (TaKaRa, Japan) and 5.7 μL of ddH₂O. DNA amplification was performed using a PTC-100 Peltier Thermal Cycler (MJ Research Inc., USA). The PCR program included denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension step at 72°C for 5 min. The PCR products were separated through electrophoresis on 8% non-denaturing polyacrylamide gels at a voltage of 180 V for approximately 100 min and then visualized via silver staining (Creste et al., 2001 (link)).

RNA was harvested using RNA Bee (Friendswood, TX, USA) according to the manufacturer’s instructions. Total RNA (5 μg) was used for cDNA synthesis. The PCR was performed using a PTC-100 Peltier thermal cycler (MJ Research, Hercules, CA, USA) in accordance with the general protocol. The cycle profile used for PCR was as follows: initial denaturation step at 95 °C for 10 min, followed by 40 cycles at 95 °C for 20 s, 60 °C for 15 s, and 72 °C for 20 s. GAPDH was used as a loading control. PCR products were electrophoretically separated on 1% agarose gel containing 0.5 μg/mL of ethidium bromide and were visualized with an ultraviolet light transilluminator. The primer sequences used are shown in Table 1.