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

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)

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
Well I hope it works.


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

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

Maddie on Jul 21 2010, 05:21 PM said:

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?

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

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

Or ordering is hell and takes forever?
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.

Maddie on Wed Jul 21 20:27:29 2010 said:

How was the presentation btw?

pito on Mon Sep 13 17:27:16 2010 said:

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...