buffer preparation calculations - (Sep/15/2008 )

Hi,

I need to make 50 ml of the following wash buffer for an Invitrogen kit:

(10 mM Tris-HCl pH7.5, 0.15M Lithium chloride, 1mM EDTA)

This what I have:

1M Tris-HCl, pH8.0
Disodium EDTA crystals (MW 372.24)
LiCl crystals (MW 42.39)

This is what I propose to do:

1. Dilute 0.5 ml of Tris-HCl with 39.5 ml of DEPC-treated water
2. Adjust pH to 7.5 with conc. HCl, keeping track of amount added
3. Make up volume to 50 ml
4. Dissolve 6.36 gm of LiCl and 372 ug of disodium EDTA and autoclave.

Are my calculations correct? Any advice would be greatly appreciated.

That would unfortunately not work, and here's why.

1- The pH of a solution should be ajusted after all the ingredients have been added, but before adjusting the final volume with water.
2- Even if the plan of using pH 8.0 Tris and ajust it at 7.5 afterwards would work, i recommend you make yourself a stock solution of Tris at pH 7.5 Will be useful for a lot of things!
3- EDTA won't dissolve unless the pH of the solution is brought to 8.0. Make a stock of 500 nM EDTA at pH 8
4- Your calculations for LiCl are not right.

You need 0,15 M in 50 ml.

1 mol LiCl = 42.39
0.15 mol LiCl = 6.36, but that is for 1000 ml
So 6.36 g for 1000 ml
x g for 50 ml
So (50 * 6.36)/1000 = 0,138 g in 50 ml

So here's what i would do :

Dissolve 0,138 g of LiCl in about 25 ml of water (no need of DEPC if RNAse are not an issue).
Add 500 µl of 1M Tris pH 7.5
Add 100 µl of 500mM EDTA pH 8.0
Adjuts volume to 50 ml.

Hope this helps!

f I understand what you are doing correctly, the calculations are not quite right

6.36 gm of LiCl and 0.372 g EDTA is the amount used to make 1000ml of solution.
Since you are weighting out EDTA powder, the EDTA is an acid and will throw off the pH calibration a little.
Both LiCl and EDTA will take up volume, the LiCl in particular. Adding these two components after topping up to 50ml will increase the volume of the solution and throw off the concentration calculations

How much of this buffer do you need?

Assuming you need only 50ml of this buffer
1 - make up 500mM EDTA, pH 8.0 solution (100ml, takes 18.61g EDTA)
2 - make up a 1000mM LiCl (100ml takes, 4.24g)

3 - add 0.5 ml of 1M Tris-HCl, pH 8.0
4 - add 0.1ml of 500mM EDTA, pH 8.0
5 - add 7.5ml of 1M LiCl
6 - add 20ml of water (of suitable purity)
7 - adjust pH
8 - add more water until until a volume of 50ml is reached
9 - weight the buffer, the glass bottle it is in and the cap. write down that mass
10- autoclave
11- weight mass again, add more sterile DEPEC water if required.

Thanks, Madrius and perneseblue, you made my week ahead so much easier. Best of luck with your own projects.