Prepare RNA extration solution

[ddong216]

Hi,could someone give me some advice?
When I prepare the RNA extraction solution,I met some problems. I need to prepare 5M KAC, ph 4.8. Every time I tried dissolving the KAc into DEPC-H20 and using the acetic acid to adjust the ph to 4.8, but it need too much acetic acid and the total volume is more than what I want. I have tried several times, but it is failed. Finally, I mixed 60ml of 5M potassium acetate(100ml) with 11.5 ml of acetic acid and 28.5 ml of DEPC-H20, this solution is 3M with respect to potassium and 5M with respect to acetate. I do not know this concentration whether is okay? And if it will affect my experimental results ?

[mchlbrmn]

The protocols I am familiar with use, I believe,
for DNA: 3/5M KAc, pH 5.2 (3M K, and 5M Acetate), or
for RNA: 2M Kac, pH 4.2
Perhaps there an error or misunderstanding in your protocol, and you are trying to do something impossible.
If you are performing the guanidinium isothiocyanate RNA preparation, the solution must have the lower pH KAc because only under acid conditions will the RNA stay in the aqueous and the DNA go into the organic phase during the extraction. I think the pH is probably more important than the exact molarity.
Also, for some solutions it is impossible to dissolve the salt completely until you also adjust the pH at the same time.

Edit: I was confused above. I meant Sodium Acetate 3M and 5M.

[ddong216]

The protocols I am familiar with use, I believe,
for DNA: 3/5M KAc, pH 5.2 (3M K, and 5M Acetate), or
for RNA: 2M Kac, pH 4.2
Perhaps there an error or misunderstanding in your protocol, and you are trying to do something impossible.
If you are performing the guanidinium isothiocyanate RNA preparation, the solution must have the lower pH KAc because only under acid conditions will the RNA stay in the aqueous and the DNA go into the organic phase during the extraction. I think the pH is probably more important than the exact molarity.
Also, for some solutions it is impossible to dissolve the salt completely until you also adjust the pH at the same time.

mchlbrmn, thank you for your replay. I use the method is SDS/phenol method, KAc forms a complex with SDS and protein. At the same time, high concentration of salts can help polysaccharide precipitation.I want to ask you a basic question, I use the method -5M KAc 100ml( I take 60ml),with 11.5 ml of acetic acid and 28.5 ml of DEPC-H20, this solution is 3M with respect to potassium and 5M with respect to acetate, ph is 4.8. I do not know the pH of this method whether is 4.8.

[mchlbrmn]

I apologize, but I think I was getting confused between sodium and potassium acetate earlier. It is sodium acetate that I use at 3M to precipitate DNA, and at 2M and more acid pH to extract RNA in the guanidinium purification (Chomzynski sp?) method. However, I think the Na and K behave similarly in regard to pH.

I think that if you have a solution of 3M potassium acetate, and 5 M acetate, that there must be one, fixed pH. However I found on the web one source saying 5.2, and another saying 4.8. I'm not at work now and don't have my information form there, so I'm not sure.

The method you describe sounds like the one I use for plasmid DNA.
As I said above, it is possible that the problem is that the salt will not all dissolve unless you start to add the acetic acid while you are still dissolving the potassium salt, and then finish adding the acid to bring it to the correct pH after the salt is completely dissolved.