Processing an thawed aqueous solution without mixing - (Dec/05/2008 )
I have a really stupid question, but it really puzzles me for a long time till now.
After thawing an aqueous solution, for example RNA dissolved in buffer, will it cause any problem if i don't mix it before processing it, like perform a RT reaction.
Sometimes i am really absent minded, just took the amount needed after i thawed them. Afterwards no matter what the data looks like i will get suspious about it.
Thanks in advance for any comments.
After thawing an aqueous solution, for example RNA dissolved in buffer, will it cause any problem if i don't mix it before processing it, like perform a RT reaction.
Sometimes i am really absent minded, just took the amount needed after i thawed them. Afterwards no matter what the data looks like i will get suspious about it.
Thanks in advance for any comments.
I am the same way. Unfortunately, I don't really have a "real" answer to your question (meaning that I have never tested it). I believe that mixing after thawing could make a difference, but this also depends on the solution (and it's contents) you are using. For example, I never use solutions until they are completely thawed because ingredients in that solution possibly freeze/thaw in a certain order, which shifts the concentration of the ingredients in the solution....some might be thawed where others might be still frozen. And then I try to remind myself to mix every solution before use (to homogenize it). I don't know how that would be for RNA in solution....if you are really interested maybe you could do a little test. Maybe there is a way to label your RNA (like ethidium bromide for DNA), unthaw it without mixing and hold it (safely) in front of a UV-light and look at the distribution....but I said, I have never tested it and I am not sure how feasible this particular method would be.
Cheers!
Yes, thawing without mixing can cause erroneous results, it is usually particularly noticeable with buffers, where the salt concentration is high, if you don't mix then your reaction may not work.
Rates of diffusion are dependent on the size of the molecule and the concentration gradient (and temperature, but we can ignore that for now assuming going from freezer to room temp) and are in the order of a 10-6 cm2 per second for aqueous solutions. So basically leaving your solution on the bench to thaw and mix will not work for any volume over a few 10s of microlitres.