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29 replies to this topic

#1 Maddie

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Posted 20 July 2010 - 07:12 AM

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?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#2 Maddie

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Posted 20 July 2010 - 07:17 AM

I can also use a 1:2.5 ratio.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#3 pito

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Posted 20 July 2010 - 07:30 AM

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.

Edited by pito, 20 July 2010 - 08:04 AM.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#4 Maddie

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Posted 20 July 2010 - 07:34 AM

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.

Edited by Maddie, 20 July 2010 - 07:57 AM.

Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#5 Maddie

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Posted 20 July 2010 - 07:36 AM

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.

Edited by Maddie, 20 July 2010 - 07:37 AM.

Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

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#6 Maddie

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Posted 20 July 2010 - 07:53 AM

Why not using 0.8 volume then?



Pito you make me doubt now :lol:
arghhhh
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#7 pito

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Posted 20 July 2010 - 07:58 AM

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..

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#8 Maddie

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Posted 20 July 2010 - 08:00 AM

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.
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#9 pito

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Posted 20 July 2010 - 08:01 AM

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.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#10 Maddie

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Posted 20 July 2010 - 08:02 AM

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.

Edited by Maddie, 20 July 2010 - 08:04 AM.

Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#11 pito

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Posted 20 July 2010 - 08:05 AM

If the ratio is most important then you could aslo try 1 volume of 4M and 2 of the extraction buffer...

(would 1 mole less of the solution matter a lot ????)

If you think it would, then go for the 0.7 ratio or maybe even 0.6

anyway: 0.7 * 4 = 2.8 in 1.5 total (if you always add up to 1.5 )
or 0.6 * 4 = 2.4 in 1.5 total (add up to 1.5)

compared to: 0.5 * 5 = 2.5 in 1.5 total.

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#12 pito

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Posted 20 July 2010 - 08:08 AM

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.



you cant change the nucleic acids , right? (I am assuming this is your DNA sample?)

So if the # of GuSCN is the most important then you need to use a bit more of the 4molar solution then when using a 5molar solution since you need to have the same amount of moles of GuSCN..

So yeah, you could use 0.7 then or even 0.6

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#13 Maddie

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Posted 20 July 2010 - 08:18 AM

Yes, the DNA is constant and the extaction buffer volume is 4ml.
So I'd need to add 2.4ml to 2.8ml of GuSCN 4M to have something similar to adding 2ml of GuSCN 5M to 4ml extraction buffer, right?
Or even better 2.5ml GuSCN 4M + 4ml extraction buffer (10 moles each time?)

But now it will be diluted by the sodium acetate ahem...

Edited by Maddie, 20 July 2010 - 08:23 AM.

Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein

#14 pito

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Posted 20 July 2010 - 08:40 AM

lol the numbers changed.

if you normally use 2 ml of 5M then you have 10 moles for 6 ml in total

so yeah, use 2.5ml of 4M so that you have 10 moles again but this time in 6.5 ml.

but do you always bneed to use 4ml extraction buffer?

Becuase if not: use less ? (take 3.5 so the end volume is also 6ml... then its less diluted, the moles per volume).

It all depends on what you want and indeed the sodium acetate.. you dolute that too then, not if you use the 3.5 in stead of 4, then it will be more: 300*2.5 = 750 in 6ml in stead of 300*2 = 600).

If you don't know it, then ask it! Better to ask and look foolish to some than not ask and stay stupid.


#15 Maddie

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Posted 20 July 2010 - 08:57 AM

OK, so if I use a stock of sodium acetate 3M, then my final concentration of GuSCN will become 3.6M right?

Then what about 3.45ml of 3.6 M GuSCN, 300 mM sodium acetate (pH 5.2) + 4ml of extract (this is my whole extract, can't change that)
3.45*3.6= 12.42 moles
12.42 / 7.45= 1.667

Close enough?
Theory is when we know everything and nothing is working. Practice is when everything is working and nobody knows why. Here, we combine theory and practice. Nothing is working and nobody knows why.

A. Einstein




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