KAPA sybergreen as a product is pretty good. Be careful to use the right amount of ROX. Have a look at this link "https://www.google.co.in/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=0CB8QFjAA&url=https%3A%2F%2Fwww.kapabiosystems.com%2Fdocument%2Fkapa-sybr-fast-abi-prism-tds%2F%3Fdl%3D1&ei=LT3KVPfeKcWqggThm4CwAg&usg=AFQjCNHabP0IuMWsSrP3wd27PS9Ze1q_bg&bvm=bv.84607526,d.eXY"
Also can we put Realtime reaction at 10ul instead 20ul reaction??
Can we play around with it?
Depends on how low your instrument can detect. Generally lowering volume sometimes also brings variations in replicates. However, you can try it out just to see if it gives you the same ct values.
The KAPA SYBR FAST 2x Master Mix has worked great for us. Our instrument is the LightCycler 480 by Roche that does not require ROX. We use the 380 well plates and 10uL reaction volumes. I have RNA-seq data that I validated by qPCR using this dye system and the degree to which the qPCR agrees with the RNA-seq data is phenomenally consistent. So, I highly recommend the KAPA SYBR FAST 2x Master Mix.
Ricardo Frausto, can you please recommend optimal reaction conditions for KAPA SYBR FAST 2x Master Mix i.e. Duration for each step?
Sanja:
You will find the reaction conditions in the attached article. Also note, I use a final primer concentration of 125nM (per primer), which has helped minimize or eliminate primer dimers.
Best.
Article Posterior Amorphous Corneal Dystrophy Is Associated with a D...
Thank you Ricardo for fast answer.
Nice publication and very helpful!
All the best,
Sanja
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