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29 replies to this topic

#16 pito

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Posted 20 July 2010 - 09:48 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?



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...

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


#17 Maddie

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Posted 20 July 2010 - 10:02 AM

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:

Edited by Maddie, 20 July 2010 - 10:04 AM.

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#18 pito

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Posted 20 July 2010 - 10:13 AM

Lol.

Yeah, you will dilute even more.

Lol

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

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...

Edited by pito, 20 July 2010 - 10:27 AM.

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#19 Maddie

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Posted 20 July 2010 - 11:26 AM

But if you add the 3M stock to the 5M GuSCN, then that isnt 5M anymore...


:D ...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. :lol:
It would have saved me a day of headache.

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

Edited by Maddie, 20 July 2010 - 11:29 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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#20 pito

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Posted 20 July 2010 - 11:35 AM

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..:lol:)

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

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#21 Maddie

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Posted 20 July 2010 - 11:44 AM

He has 5/3=1.666666
and I have 12.42/7.45= 1.6671
(4ml extract + 3.45ml of my Guanidium + sodium acetate mixture)
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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#22 Maddie

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Posted 20 July 2010 - 11:52 AM

Ahaaaaa...1 more thing. He said 1:2 but also 1:2.5 worked good.
With 1:2.5, you go down to 1.428

With 3ml at 3.6M, I am at 1.54 which is still between 1.6 and 1.4

YEAHH :lol:
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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#23 pito

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Posted 20 July 2010 - 12:25 PM

Lol
Well I hope it works.


PS. why does it always have to be 4ml of extraction buffer?

Edited by pito, 20 July 2010 - 12:25 PM.

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#24 Maddie

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Posted 21 July 2010 - 07:21 AM

This is the amount of buffer (EDTA) necessary to decalcify 200mg of bone powder entirely.
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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#25 pito

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Posted 21 July 2010 - 07:23 AM

This is the amount of buffer (EDTA) necessary to decalcify 200mg of bone powder entirely.


But cant you adjust it a bit?
I mean: it seems harsh to state you always need 4ml? There must me some margin on it?

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#26 Maddie

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Posted 21 July 2010 - 07:43 AM

Oh yes there is. Basically you need to respect a certain ratio EDTA/ bone powder to demineralize the whole sample. So the less powder, the less buffer. This week and next, I will be comparing my colleague's protocol with mine and I already have decalcified 4 extracts with 4ml.
However, if I decide to test other samples, then I can adjust to 3.5ml (although I'm still hoping to receive powdered GuSCN before Thanksgiving <_< ).
Of course using 3.5ml would make me do the calculations all over again :D
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

#27 pito

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Posted 21 July 2010 - 09:35 AM

Well I hope it all works out fine.
Is the GuSCN powder that expensive then or?

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#28 Maddie

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Posted 21 July 2010 - 12:27 PM

Or ordering is hell and takes forever? :lol:
Did I mention I'm supposed to present data from this at a conference in the begining of September. This explains my hurry ;) .
Stress, stress.
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

#29 pito

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Posted 13 September 2010 - 09:27 AM

Or ordering is hell and takes forever? :lol:
Did I mention I'm supposed to present data from this at a conference in the begining of September. This explains my hurry ;) .
Stress, stress.


How was the presentation btw?

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#30 Maddie

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Posted 15 September 2010 - 06:22 AM


Or ordering is hell and takes forever? :lol:
Did I mention I'm supposed to present data from this at a conference in the begining of September. This explains my hurry ;) .
Stress, stress.


How was the presentation btw?


Well...it went OK I guess except that I presented what I WANTED to do instead of what I had done...which sucks.
My extractions rocked though. The buffer worked very well and the purity of the extract is far better than what I had before.
Now I have DNA clean enough to make next generation sequencing libraries. Moving slowly, but moving...
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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