# I got a though one - Buffer preparation (Jul/20/2010 )

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OK, I need to prepare the following solution: 5 M GuSCN, 300 mM sodium acetate (pH 5.2).
Then I should mix 1 volume of this solution with 2 volumes of extraction buffer.
My problem is that I only have 4M Guanidium thiocyanate (liquid). My colleague told me to use 0.7 volume of a solution prepared with the 4M GuSCN instead of 0.5 volume with the 5M GuSCN when I mix with the extraction bufer.
What do you think?

I can also use a 1:2.5 ratio.

0.7 * 4 = 2.8 in a total of 1.5

0.5 * 5 = 2.5 in a total of 1.5..

(I assumed you will use 0.5 volumes (or 0.7) and add 1.0 volume (or 1.4) so having a total of 1.5 ).

Why not using 0.8 volume then?

Or is it important to have 2 times more buffer ?

It all depends on how important the buffer is.

-pito-

Hmm here is what I got 5M/3( 2 volumes of extraction buffer+ 1 volume of buffer)= 1.6666
4/1.666= 2.4
So what about 1:1.4? wouldn't that work?
That would actually be the 0.7:1 my colleague suggested.

pito on Jul 20 2010, 11:30 AM said:

Or is it important to have 2 times more buffer ?

My colleague optimized the ratio and 1: 2 or 1:2.5 was the best.

pito on Jul 20 2010, 11:30 AM said:

Why not using 0.8 volume then?

Pito you make me doubt now
arghhhh

Well its simple: you can never obtain the same ratios ..

If you start with a 5molar solution you can either: make sure you have a 1/2 ratio with the extraction buffer and then have less of the solution.

Or you can have the same amount of solution (by using more of it) but then you have not a 1/2 ratio...

I do not know what the best is...

If the ratio is more important, then go for the ratio..

-pito-

yes, the ratio is the most important. So which one is best?
0.8 to 1 or 0.7 to 1? I am totally lost now.

if you use 0.8 with 1.2 then you do not have a 1/2 ratio...

if you use 0.7 and 1.3 then you also do not have a 1/2 ratio, but its closer...

you just need to find out whats the most imporant one: having enough of moles of the solution or having the correct ratio.

-pito-

Sorry, I meant the ratio between molecules (moles?) of GuSCN and nucleic acids needs to be the same.
I need to have the right amount of salt, so that my DNA will bind properly to the silica beads.