In several experiments we determined the levels of Xist by RT-PCR. For experiments with ActinomcyinD treatment, the drug was dissolved in DMSO at 1mg/mL and added to the culture medium to a final concentration of 1μg/mL. For RT-PCR, cells were harvested in 1mL TRIzol (Thermo Fisher), after culture medium removal and PBS wash. RNA was purified over RNAeasy columns (Qiagen). 1μg total RNA was used in a reverse-transcription (RT) reaction with SuperScript III and appropriate strand-specific reverse primer, according to manufacturer’s instructions (ThermoFisher). 1/20th of the RT reaction was used in a quantitative PCR reaction, using either 480 SYBR Green LightCycler PCR mix (Roche), SsoAdvanced Universal SYBR mix (Bio-Rad) or SYBR Green Master Mix (Applied Biosystems) and appropriate primers (see Supplementary Table 1 ), in triplicate reactions. RT-qPCR experiments were normalized against Gapdh or Rrm2 transcripts.
480 sybr green lightcycler pcr mix
Manufactured by Roche
The 480 SYBR Green LightCycler PCR mix is a ready-to-use solution for real-time PCR amplification and detection using the SYBR Green dye. It is designed for use with the Roche LightCycler 480 System.
Automatically generated - may contain errors
2 protocols using 480 sybr green lightcycler pcr mix
1
Quantification of Xist Transcript Levels
2
Quantification of Xist Transcript Levels
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In several experiments we determined the levels of Xist by RT-PCR. For experiments with ActinomcyinD treatment, the drug was dissolved in DMSO at 1mg/mL and added to the culture medium to a final concentration of 1μg/mL. For RT-PCR, cells were harvested in 1mL TRIzol (Thermo Fisher), after culture medium removal and PBS wash. RNA was purified over RNAeasy columns (Qiagen). 1μg total RNA was used in a reverse-transcription (RT) reaction with SuperScript III and appropriate strand-specific reverse primer, according to manufacturer’s instructions (ThermoFisher). 1/20th of the RT reaction was used in a quantitative PCR reaction, using either 480 SYBR Green LightCycler PCR mix (Roche), SsoAdvanced Universal SYBR mix (Bio-Rad) or SYBR Green Master Mix (Applied Biosystems) and appropriate primers (see Supplementary Table 1 ), in triplicate reactions. RT-qPCR experiments were normalized against Gapdh or Rrm2 transcripts.
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!