Primers were designed for randomly selected 7 DEGs from Trombay wild CDS with the help of Gene Runner v. 5.0.99 Beta software (Hastings Software, Inc.).Total RNA extraction from both Trombay wild and TU94–2 variety were performed with Spectrum™ Plant Total RNA Kit (Sigma-Aldrich, USA) and treated with DNAse-I (Sigma-Aldrich, USA) to eliminate traces of genomic DNA. The cDNA was synthesized using a PrimeScript™ RT-PCR Kit (Clontech, USA) and real time PCR protocol was followed according to manufacturer’s instructions given in SYBR1 Premix ExTaq™ (Tli RNAse H Plus) (Clontech,USA). The thermal cycling conditions were carried out in Rotor-Gene-Q Real-Time PCR System (Qiagen, USA) using the following program- 95 °C for 5 min followed by 40 cycles of 94 °C for 30 s, 62 °C for 20 s and 72 °C for 20 s followed by melting PCR products from 65 °C to 95 °C. The relative gene expression levels were calculated by REST software [98 (link)].
Sybr1 premix extaq tli rnase h plus
Manufactured by Takara Bio
Sourced in United States
SYBR1 Premix ExTaq™ (Tli RNAse H Plus) is a ready-to-use master mix for real-time PCR amplification. It contains SYBR Green I dye, Tli RNase H Plus DNA polymerase, and other necessary components for efficient and specific DNA amplification.
Automatically generated - may contain errors
2 protocols using sybr1 premix extaq tli rnase h plus
1
RT-qPCR Gene Expression Analysis
2
RNA Extraction and qRT-PCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from immature seeds of Trombay wild and TU94-2 plants with the help of Spectrum Plant Total RNA Kit (Sigma-Aldrich, USA) and treated with DNAse-I (Sigma-Aldrich, USA) to eliminate traces of genomic DNA. The cDNA first strand synthesis was done using a PrimeScript RT-PCR Kit (Clontech, USA) and quantitative real time PCR was performed following manufacturer's instructions given in SYBR1 Premix ExTaq (TliRNAse H Plus) (Clontech,USA). The PCR amplification was carried out in Rotor-Gene-Q Real-Time PCR System (Qiagen, USA) with the following program, 95˚C for 5 min followed by 35 cycles of 94˚C for 30 sec, 62˚C for 20 sec and 72˚C for 20 sec followed by melting of PCR products from 65˚C to 95˚C. Quantitative real-time PCR experiments for all gene-specific primers were performed twice with three biological replicates run in triplicate. The relative gene expression levels were calculated by relative quantification (RQ) through the 2 -
ΔΔCt method [33] .
ΔΔCt method [33] .
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!