Phenol/Chlo/EtOH

Barry Moore barrym at stella.med.utah.edu
Fri Jun 9 11:12:59 EST 1995


Greetings,

I am working on a DNA cloning project that requires a series of enzymatic reactions
each followed by extraction of my DNA with Phenol/Chlorofom and percipitation with
EtOH.  According to Maniatis and others I should expect to lose between 5%-10% of my
DNA in each extraction-percipitation cycle, but I have been losing  more than that.
I have asked all the other workers in the surrounding labs, and no one else seems
to have ever had this problem, so I thought I would go to a larger audience for help.
I am going to describe my technique as breifly as possible, and I would appreciate any
comments or suggestions that anyone has on where my problem might be.

20 ul DNA (approx 300ng/ul)

Add 1 volume from lower phase in Phenol/Chloroform/Isoamyl mixture
Vortex breifly (the DNA is 8-40 kb in different samples)
Spin 12,000 rpm for 1 min at room temp.
Remove top layer

Add 20 ul H2O to phenol phase and repeat the above (back extract the phenol).
Combine the two aqueous (top) phases

Add 1 volume of chloroform
Vortex breifly
Spin at 12,000 rpms about 1 min.
Remove top layer

Add 1/10th volume of 3M NaAc (pH 5.2)
Add 2.5 volumes absolute EtOH
-20 C for 15-30 min.
Spin 12,000 rpm in cold room for 15 min.
Pour off EtOH (I've never seen a visible pellet come off at this step)

Add approx. 1 ml of 70% EtOh
Spin in cold for 15 min.
Pour off 70% EtOH
Speed Vac for 20 min.

Resuspend in 20 ul.

Anybody see anything that they do differently?

Thanks for your help.

Barry


--

**************************************************
*                  Barry Moore                   *
*               Univeristy of Utah               *
*           Department of Human Genetics         *
*            Salt Lake City, UT 84123            *
*                     USA                        *
*                (801) 281-4636                  *
*            barrym at corona.med.utah.edu          *
*    http://www-genetics.med.utah.edu/~barrym    *
**************************************************



More information about the Methods mailing list