CTAB extraction problem - CTAB extraction problem (Aug/08/2011 )

Hello,

I am not experienced in molecular biology procedures and have encountered some problems in a CTAB DNA extraction in two plant species from the Lauracea family.

When I add the CTAB buffer to the powder, the mixture forms a very viscous substance. The main problem in that after I add the cloroform/isoamylalcohol (24:1) solution and spin at maximum speed, literature says that it is expected that I obtain a three layer fase in the tube; the top fase is the aqueous solution with the DNA and what I should retain (supernatant), followed by a layer with the cloroform/isoamylalcohol and followed by the cell debris at the bottom of the tube.

What I obtain is something quite diferent; the supernatant fase is usually very little and sometimes I don´t obtain any supernatant, followed the cell debris fase and at the bottom of the tube an aqueous layer which I expect should be the cloroform/isoamylalcohol.

Can anybody tell me what can be the problem here. Why don´t I get supernatant and why does the cell debris stay in the middle of twi liquid fases?

Thanks in advance for any help

Carlos

The middle layer is typically the cell debris that has not dissolved. You probably need to grind your samples into a powder to get this to work properly, use liquid nitrogen or dry ice to do this. Viscosity may indicate that the DNA has been extracted and is clogging up the mix.

You may need to do your extractions in a bigger volume as well.

Yeah...sounds like you need more volume, or a smaller sample size.