I am using Sigma DNase I (AMPD1) where I add 1 ul of each component(DNAse1, buffer and stop solution) to 8 ul of RNA in water (below is the link to the protocol)
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/ampd1bul.pdf
According to the protocol, it did not really state how much of the total RNA that I need to put in the 8 ul of water so I am a little confused now.
Help appreciated!! Thanks
According to the Sigma bulletin that you linked:
One unit of DNase I, Amplification Grade, completely digests 1 ug of plasmid DNA to oligonucleotides in 10 minutes at 37 °C.
As long as your total DNA content is less than 1ug you should be ok with just 1uL. You can also incubate for longer than 10 minutes to make sure the digest is complete. I usually use the nanodrop to quantify beforehands (know that it cannot distinguish RNA and DNA so I take the total and use a corresponding amount of DNAse). To more accurately quantify RNA you should use qubit or picogreen fluorimetry.
You need to add the buffer and DNAseI first, then after incubating at 37 degrees you must add the stop solution.
Thank you so much for your answer. I also routinely check my RNA yield before going for further procedures. From your answer, I take that it should be okay to just add 1 ul of DNAse I and buffer each to my total RNA extract (20ul), then quantify them again?
- Badges
- Science method