t.barnett at path.utas.edu.au
Tue Feb 20 19:05:58 EST 1996
In article <stebbin1-190296161740 at triezenberg2.bch.msu.edu>, stebbin1 at pilot.msu.edu (John Stebbins) wrote:
> We use Qiaex II from Qiagen to isolate DNA fragments from agarose for the
> purposes of ligation. We have had LOTS of problems getting our ligations
> to work. Has anybody else had similiar problems? Has anybody figured out
> how to correct the problem. I realize one correction would be to use a
> different method to isolate DNA from agarose. Anybody have a favorite
> John Stebbins
I have had a lot of trouble with the quiex procedure as well. I now extract DNA from LMP agarose using a simple phenol/chloroform procedure.
1. Excise band from LMP agarose
2. Melt for 5min at 70C
3. Cool for 30s at RT and add an equal volume of TE saturated phenol
4. Spin for 5min in a microfuge
5. Remove aqueous phase and re-extract with an equal volume of phenol
6. Extract with phenol/chloroform and ethanol ppt.
Yeilds aren't great, but this method works well for me.
More information about the Methods