Total RNA was extracted by the Trizol method and reverse transcribed according to the PrimeScript™ Real-time Master Mix kit (Takara, Beijing, China). We took 1 μL each of PEDV-F and PEDV-R primers, 1 μL of the reverse transcription product, 12.5 μL of 2×Mix, and 9.5 μL of ddH2O for a total volume of 25 μL. The reaction conditions were 95 °C for 5 min, 95 °C for 30 s, 58 °C for 30 s, 72 °C for 1 min, 35 cycles, and 72 °C for 10 min. The PCR products were identified by 1.0% agarose gel electrophoresis. PEDV-specific primer sequences are as follows (Forward: 3′-TAGGACTCGTACTGAGGGTGT-5′; Reverse: 3′-CTATTTTCGCCCTTGGGAATT-5′).
Primescript real time master mix kit
Manufactured by Takara Bio
Sourced in China
The PrimeScript™ Real-time Master Mix kit is a ready-to-use reagent for performing real-time PCR (RT-PCR) experiments. It contains all the necessary components, including a reverse transcriptase enzyme and a DNA polymerase, to facilitate the amplification and detection of target RNA sequences.
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Lab products found in correlation
2 protocols using primescript real time master mix kit
1
PEDV Detection by Real-Time RT-PCR
2
Quantitative Gene Expression Analysis
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Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) according to the manufacturer’s instructions. For cDNA preparation, the PrimeScript Real-Time Master Mix Kit (Takara) was used according to the manufacturer’s instructions. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) was performed on a real-time detection system using the iTaq Universal SYBR Green Supermix Kit (Bio-Rad). PCR conditions included 1 cycle of 95°C for 3 min and 40 cycles of 95°C for 5 s and 60°C for 30 s. The housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a normalization control. A list of the primer used is provided in table S1.
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