4 replies to this topic

[jamestoon1]

My DNA and RNA isolation kits require me to dilute some of the buffers with absolute ethanol.
I added denatured ethanol to all the necessary buffers, then only I came to realize that we should use non-denatured ethanol for molecular biology applications because denatured ethanol contains impurities.
What should I do now?
Can I proceed with DNA and RNA isolation using the reagents?
If not, my supervisor is going to kill me OMG!!!

[phage434]

Denatured ethanol typically has some high molecular weight compounds that are relatively hard to evaporate. They will probably work well until it comes time to evaporate the wash. The evaporation may leave some organics that are hard to remove. Most likely the buffers you are adding ethanol to are simple Tris buffers with 70% or 80% ethanol, and can be easily replaced. See PE buffer, for example:
http://openwetware.o.../Qiagen_Buffers

[hobglobin]

But the denatured ethanol with the molecular compound (e.g. butanone or denatonium) is an azeotrope and both this mixture will evaporate as it is a pure liquid. I think the problem is that these used compounds (or other impurities) may interfere with DNA. Anyway there are labs that work with denatured ethanol for washing purposes and have no problems (I won't do it).

[phage434]

The situation is complicated, since there are several variations on denaturants, often country specific, and application specific.

[jamestoon1]

Thanks for all the replies.
Due to shortage of time (I need to do some trial tests today), I proceeded with DNA and RNA isolation with the reagents added with denatured ethanol.
The spectrophotometric readings were good - the yield and purity were comparable to DNA/RNA isolated with the reagents with non-denatured ethanol.
Let's hope any impurities that may be present would not interfere with downstream applications.