[shivaol88]

Hi,
Just having an issue with making up a solution and don't trust myself that i am doing it properly!

My protocol says that i need to prepare 0.05M Tris HCL buffer PH 7.4, containing 1.0M NACL, 20mM EDTA, 1mM PMSF.

I need to make 3ml in total.

I have 1M [font="""]stock[/font] solution of Tris HCL buffer PH 7.4, and EDTA, NACL, and PMSF in solid form.

I am thinking that I probably do not need the [font="""]NACL[/font] as its purpose was probably to adjust the PH to the desired level but as mine is already at 7.4 i might not need it.

Does anybody know the right quantities to make up this solution or can offer any advice in this area i would greatly appreciate it.

Thanks

Siobhan

[Greippi]

I have never heard of NaCl being used to adjust pH, if you're using pure NaCl then it definitely shouldn't affect pH. I mean, Cl- can act as a weak acid, and Na+ can be a weak base, but the effects in a solution like that are negligible. What is the solution for? The NaCl is most likely a vital ingredient, especially as it's in there at a not insignificant concentration!
For pH-ing something like that I'd use HCl or NaOH. It's probably best to make up more than 3ml of your solution, as it's quite difficult to make up something like that in small volumes, especially as you have to pH it.

So what, exactly are you stuck on when it comes to making up the solution? Do you know how to make up a solution of a certain molarity? Do you know your equations? Or are concentration calculations something you've never come across.

This post has been edited by Greippi: 11 January 2012 - 04:32 PM

[Greg Boyles]

shivaol88, on 11 January 2012 - 03:01 PM, said:

Hi,
Just having an issue with making up a solution and don't trust myself that i am doing it properly!

My protocol says that i need to prepare 0.05M Tris HCL buffer PH 7.4, containing 1.0M NACL, 20mM EDTA, 1mM PMSF.

I need to make 3ml in total.

I have 1M [font="""]stock[/font] solution of Tris HCL buffer PH 7.4, and EDTA, NACL, and PMSF in solid form.

I am thinking that I probably do not need the [font="""]NACL[/font] as its purpose was probably to adjust the PH to the desired level but as mine is already at 7.4 i might not need it.

Does anybody know the right quantities to make up this solution or can offer any advice in this area i would greatly appreciate it.

Thanks

Siobhan

0.05M is simply 1/20 the concentration of 1M

So you would take 5ml of your stock and mix it with 95ml of water.....do that x times to end up with the volume you require.

If you need the NaCl then work out the total weight required for the total volume of your working solution and just dissolve it in the working solution.

This post has been edited by Greg Boyles: 11 January 2012 - 04:55 PM

[CharonY]

For biological buffers NaCl is usually there for osmotic stability. Obviously Tris-HCl is adjusted by adding HCl (hint in the name) as Tris is a base. You have to be careful, though, as with Tris pH is also concentration-dependent. Strong dilutions will show a pH shift. For the rest of the salts you have to calculate the weight of the salts for a given volume that you are going to prepare to obtain the desired molarity.