How to prepare a "home-made" lysis solution compatible with RNA extracti - (Aug/26/2014 )

Hi everyone,

I am using a kit to extract total RNA.

I want to prepare a lysis solution compatible with the columns in the kit.

I know that the lysis solution contains GITC (guanidium thiocyanate), so I tried to do by myself this solution :

- test 1 : GITC 4M; Sodium citrate pH 7 - 25 mM; sarkosyl 0,5%; beta-mercapto 1%

- test 2 : GITC 4M; Tris-HCl pH 7,6 - 25 mM; NaCl 100 mM; EDTA 10 mM; sarkosyl 0,5%; beta-mercapto 1%

I apply the lysis solution on the plant tissue powder, I add chloroform:IAA to obtain a clear supernatant. Then, I apply this one on the first column (filtering column).

After that, I add "binding solution" to the filtrate and the problem is here !! After mixing the filtrate and binding solution, there is a precipitation (which does not occur when I use the lysis solution from the kit).

If I apply this precipitation reaction on the next column (binding column) and I finish the protocol, I extract nothing.

So if everyone have a suggestion to modify my lysis solution or for add a step in the protocol, that would be great !! Thanks.