[whywhywhy]

the protocol for the lysis buffer i use is:

5mM Tris
1mM EDTA pH7.4

does it mean that the lysis buffer has to be pH7.4 or does it mean that the EDTA buffer alone is ph7.4? can you guys tell me which one of these options is correct?

1. add EDTA and Tris to the same bottle with the correct amounts and adjust the pH to 7.4. the lysis buffer will be pH7.4


2. make a stock solution of EDTA pH7.4 and add the right volume to the Tris solution. the lysis buffer won't be pH7.4 (due to the Tris)

i appreciate for any ideas!
thanks alot!

[AWK]

Probably it means that concentrations of  EDTA iin  final solution is 1 mM, Tris - 5mM, and pH is adjusted to 7.4.
So you should prepare solution of 2 mM EDTA and  10 mM Tris and mix them in ratio 1:1 (V/V) and then correct pH.

[whywhywhy]

hi AWK,

thanks for your reply, but i am still VERY confused.


so in this case the lysis buffer has to be at 7.4? how can we know when a buffer is a main component in a solution (like in this case, EDTA is the main component since the lysis buffer is itself) and when it is just a part of a solution? for instance the protocol for the Sample buffer shows this:

3.8ml dH20
1.0ml 0.5M Tris-HCl, pH 6.8
0.8ml glyserol
10% (w/v) SDS
1.6ml B-mercaptoethanol
0.4ml 0.1% (w/v) Bromophenol blue.


we can see here that Tris-HCl is just a part of the solution. the pH and the concentration here is definetely not at its 6.8 and o.5M like the above example. if i hide the volumes of the components, we would think that this buffer has to be at 6.8 and the Tris-HCl has a final concentration of 0.5M.
since the above example does not mention the volumes, do you think that EDTA is just a part of the solution (option 2) ?

i hope for replies, since i am going insane now!

thanks alot!

[AWK]

Generally, buffer solution do not change pH after dilution (frankly saing, the change of pH cna be neglected, but exists). Multicomponent biochemical solutions contain many reagents, some of then can change pH indeed.
EDTA is used as disodium salt and is slightly alkaline, SDS (sodium dodecyl sulfate) is nearly neutral. So after mixing, the solution may show pH not exactly equal to needed value, but usually close to it. Then biochemists adjust pH (under control of pH-meter) to needed value,
usually by addition of small volumes of NaOH or HCl solution. This is absolutely normal procedure, especially for bulk solution.
I your case, when the final volume is quite small, you should believe in the pH value, or use pH-stat system.