I want to perform in vitro transcription of large fragments (>10kb). I am concerned about the stability of my mRNA strands so I want to avoid degradation at all costs. I am using the mMessage mMachine SP6 kit for my transcription. Should I use sigma water or DEPC treated water for my transcription?
Hi Jeroen,
DEPC treated water, if not prepared very accurately (you have to make sure the all the diethyl pyrocarbonate has been removed!), will risk inhibiting the transcription reaction by also affecting the SP6 polymerase. My suggestion would be to use water taken directly from a MilliQ purification system in RNAse free tubes, if you have this possibility. The MilliQ water goes through an active carbon filter and 2-3 ion exchange columns and in the end is UV irradiated, so hardly any biological material will come out and if it will it will not be intact or functional. In my experience it's the safest and most efficient solution.
The Sigma molecular biology water, if is not the DEPC-treated one, is just filtered with a 0.1 um filter, but probably is produced by a MilliQ system to start with, since now Sigma is part of Merck/Millipore...
Good luck!
I also prefer Sigma water. RNases are omnipresent. It is good to perform RNA work as much as possible under RNase-free conditions.
I always used ultrapure water and never had any issue.
https://www.thermofisher.com/order/catalog/product/10977023
I agree with all previous answers.
The key is to maintain the RNase/DNase grade of the water. To do that in my lab we aliquot the Sigma/Fisher RNAse/DNAse free water stock into small aliquots inside a cell culture hood. This way you are sure every aliquot is as good as the original and avoid contaminating the water stock during the experiments (50 ml of water stock can be used for many experiments).
Best!
The most important think is to use RNAse free water, and eppendorff tubes RNAse and DNAse free. Sigma water is good, also Thermofisher water or milliQ+ water. If you still have degradation problems, you may use an RNAse inhibitor like Applied Biosystems one.
Sigma, Thermo etc all sell DNAse/RNAse free water. Never mind for the brand. Also the tubes should be certified. If I need to make large quantities of buffers e.g. for in situ hybridisation, I prepare DEPC-treated water for this purpose (attention harmful, has to be autoclaved after overnight incubation).
- Badges
- Science topic