Why is EB added into RNA loading buffer? - (Sep/28/2006 )
I have purchased the PROMEGA's in vitro transcription kit.
In the Protocol, there is saying that ethidium bromide was one the components of RNA loading buffer.
Why? Why not to add EB into the agarose gel to make RNA seen?
And in the Molecular Cloning 3rd edition, there wasn't EB in any of the loading buffers.
To be honest, I don't like the idea of EB in my loading buffer at all. By the time it sits around on the bench for weeks it won't be doing much anyway. They probably use it because it helps disassemble proteins? I couldn't be sure, I can't imagine them using enough in their loading buffer to matter at all.
EB works by intercalating between base pairs. Since RNA is single stranded you would think EB wouldn't work to visualize RNA. However, RNA does form hairpins and other secondary structure that is double stranded, so EB can intercalate between those base pairs. So for the small amounts of RNA produced by your kit, simply adding EB to the gel may not be enough to visualize it.
I've run quite a few samples with the same kit and my own loading buffer. It didn't make much of a difference for me... I just added it to the gel as usual.
Thanks for your advises:)