Making a Buffer - how do this (Dec/06/2006 )

How calculate 2 liters buffer w/ 25mM Tris-Base, 3.5mM SDS, 20% Methanol, pH 8.3

Reagents;
Sodium dodecyl sulfate (SDS) 288.4g
CH3OH 32.04g
HCL 36.46g
NaOH 40g


First calculate mg for Tris and SDS
ex.
Tris 2L(25mmol/1L)(.211mg/1mmol)= 6.055mg

SDS 2L(3.5mmol/1L)(.2884mg/1mmol)= 1.016mg

BUT THEN WHAT ?????

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Recalculate your tris and SDS amounts. Formula Weight of tris base is 121.14, not 211. Also, your mg calculations are incorrect, for example, SDS is 0.2884 g/mmol, not mg/mmol

20% Methanol: this is just 20% in volume vs volume of your buffer (400ml in 2L)

Combine your reagents in 2L and check the pH. Add HCl or NaOH (be careful, sometimes the pH can change quickly) until you reach a pH of 8.3.

Edit: if you are still unsure, find someone in your lab to help you. It's better to bug someone than to make your reagents wrong.

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tris base 25 mM
mW = 121.14
so, to prepare 2 liters ta 25 mM
you need : 6.057g

because for 1L at 1M you need 121.14g
for 2L you would need 121.14*2
for 0.025M you would need 121.14*2*.025

SDS
MW = 288.4 g
you need 288.4*2*0.0035 = 2.01g

methanol 20%
you need 20 mL per 100mL of solution
so you need 400 mL

so you weight 6.057 g of tris, you add water up to around 1L, then with the help of a pH-meter you add concentrated HCl until the pH goes down to pH8.3.
then you add 2.01g of SDS, you add water up to 1600mL and you add 400 mL of methanol.

and now you have your buffer.

(be careful while weighting the SDS, wear a mask, it's irritating)

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