CMK cells were transfected with the indicated siRNA. Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Valencia, CA). cDNA was synthesized using Oligo(dT)-primers with SuperScript-First-Strand Synthesis kit (Invitrogen, Carlsbad, CA). cDNA was amplified with Opticon-3 thermal cycler (MJ Research, Waltham, MA) using 2xSYBR Green (Invitrogen). Primers used were as follows: JAK3 Forward: 5′-GGCCCCATCACTCTGGACT-3′, JAK3 Reverse: 5′-GCCCTTATAATCAGGACCAAGG-3′, IL2Rγ Forward: 5′-CCACAGCTGATTTCTTCCTGA-3′; IL2Rγ Reverse: 5′-GCAGAGTGAGGTTGGTAGGC-3′, GAPDH Forward: 5′-TCCTGCACCACCAACTGCTTAG-3′, and GAPDH Reverse: 5′-GGCATGGACTGTGGTCATGAG-3′.
Opticon 3 thermal cycler
Manufactured by Bio-Rad
Sourced in United States
The Opticon-3 thermal cycler is a laboratory instrument designed for the amplification of DNA or RNA samples. It features a peltier-based temperature control system to precisely regulate the temperature of samples during the thermal cycling process. The Opticon-3 supports a range of sample volumes and can accommodate a variety of reaction tube formats.
Automatically generated - may contain errors
3 protocols using opticon 3 thermal cycler
1
JAK3 and IL2Rγ Expression Analysis in CMK Cells
2
Quantifying Target Gene in gDNA using qPCR
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The quantity of the target gene in the gDNA was estimated using the qPCR method. The amplification reactions contained 0.5 μM nested primer pairs (Table 1 ), the DyNAmo HS SYBR Green master mix (Finnzyme, Espoo, Finland), and 15–25 ng gDNA or 5 μl of the serially diluted standard DNA. The qPCR assays were performed using an Opticon 3 thermal cycler (MJ Research, Waltham, MA, USA) with the following program: 95°C for 15 min, 40 cycles of 95°C for 10 s, 58°C for 20 s, and 72°C for 30 s. The melting curve analysis was conducted by serially increasing the temperature at a rate of 0.2°C per 1 s from 45°C to 95°C. The copy number of each standard DNA sample was calculated from the amount and molecular mass of the linearized plasmid using a DNA molecular weight calculator (http://www.currentprotocols.com/WileyCDA/CurPro3Tool/toolId-8.html ). The Ct values were determined using the Opticon Monitor Software (MJ Research). The standard curve of the Ct value vs. the copy number was generated and used to calculate the total number of genome in the target gDNA template. The experiments were repeated six times, and each repetition included two technical replicates.
3
JAK3 and IL2Rγ Expression Analysis in CMK Cells
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CMK cells were transfected with the indicated siRNA. Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Valencia, CA). cDNA was synthesized using Oligo(dT)-primers with SuperScript-First-Strand Synthesis kit (Invitrogen, Carlsbad, CA). cDNA was amplified with Opticon-3 thermal cycler (MJ Research, Waltham, MA) using 2xSYBR Green (Invitrogen). Primers used were as follows: JAK3 Forward: 5′-GGCCCCATCACTCTGGACT-3′, JAK3 Reverse: 5′-GCCCTTATAATCAGGACCAAGG-3′, IL2Rγ Forward: 5′-CCACAGCTGATTTCTTCCTGA-3′; IL2Rγ Reverse: 5′-GCAGAGTGAGGTTGGTAGGC-3′, GAPDH Forward: 5′-TCCTGCACCACCAACTGCTTAG-3′, and GAPDH Reverse: 5′-GGCATGGACTGTGGTCATGAG-3′.
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