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

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.

Maddie on Jul 20 2010, 06:02 PM said:

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

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...

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).

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?

Maddie on Jul 20 2010, 06:57 PM said:

wow I do not understand what you mean with: OK, so if I use a stock of sodium acetate 3M, then my final concentration of GuSCN will become 3.6M right?
How can you go from 3M to 3.6M ?

I am getting confused now.

I was under the assumption that you had a 4M solution of GuSCN that contains 300mM of sodium acetate...

The stock of sodium acetate is 3M and I need 300mM so I do a 1/10 dilution.
If I ad for ex 1ml to 9ml of GuSCN, then the GuSCN will change to 3.6M, no?
The sodium acetate isn't in my 4M GuSCN yet..so I dilute even more

Lol.

Yeah, you will dilute even more.

Lol

I was under the assumption you had the sodium acetate in your GuSCN solution

But to be honest, I do not follow anymore ..

If I understand it correct you should start with 5M GuSCN , but with how much sodium acetate would you dilute this??
Because you speak of 3M acetate (stock) and you need 0.3 ?
But if you add the 3M stock to the 5M GuSCN, then that isnt 5M anymore...

Or should it be like this: 5M GuSCN with 0.3M acetate?

if this is true then you needed a stock solution of lets say 6M or more GuSCN , since you dilute this with the sodium acetate...

pito on Jul 20 2010, 02:13 PM said:

...excellent question Pito.

The article says: 5 M GuSCN and 300 mM sodium acetate (pH 5.2).

I think it is MUCH easier when you start with powdered GuSCN
Which is what the author of the paper recommends.
Too bad I don't have any.
It would have saved me a day of headache.

So what do you think of my 3.45ml of GuSCN 3.6M?

Lol, yes, you need to start with powder.

Anyway: if you start with what you have, it will never ....NEVER work .. lol
You can only try and get +- the same ..

9 liter of 4M = 36 moles for 9 liters

if you then add 1liter of sodium ac. its 36 moles for 10 liters so yeah, 3.6M

(ok I use liters here not ml, but its the same..)

Anyway: it will never be what you should have (according the article) you can only try to have +- similar quantities