Clean-up DNA for ligation

lees at afrc.ac.uk lees at afrc.ac.uk
Sun Jul 18 13:46:00 EST 1993


Mary Metzler, Plants, x8076 (METZLER at ALA.BTK.UTU.FI) wrote:
: Has anyone tried Gelase, which is an enzyme which digests LM agarose,
: which eliminates the necessity of using Geneclean? I am tired of the
: low yields and unreliability of Geneclean. I also sometimes have to
: work with big fragments which aren't compatable with Geneclean. Has
: anyone used Gelase? You are supposed to be able to simply ethanol
: precipitate the DNA in your gel slice after digesting with this
: enzyme for an hour. Does anyone know if it works, and if it is
: expensive?
: Thanks,
: 	Mary Metzler
: 	University of Turku
: 	Turku, Finland
: 	Metzler at sara.cc.utu.fi
 
 Recently, I have stopped using all these methods. I use a modification of
a method that was posted here recently . I cut and load about 20% more then
I need. Run the gel, cut out the band of interest with a minimum agarose.
Put the slice in a PCR tube which contains some glass fibers,
and freeze (liquid N , or Dry ice). Make a hole
with 25g needle (I make it bottom AND lid). Put tube into a 1.5ml tube and
spin (I do 15min' at least). The DNA is at the 15ml tube nice and clear.
 
Since it is very easy, and yields are good and reproducible, I now use it
regularly.
 
BTW, the original method posted, also used a special buffer to be added with
the band. I ommit this one. I don't know if that affects the yield.
 
Hope this helps. Benny.
 **************************************************************
* Benny Shomer                                                 *
* Tel-Aviv University                                          *
* Sackler School of Medicine, Dept.of Embryology and Teratology*
 
Is there a need to clean up the DNA sup after spinning? I always do, for fear of
those much talked about inhibitors in agarose.  Usually I do a phenol/
chloroform step then ETOH ppt, but I'd like to omit these steps if possible
I tend to loose too much DNA if the fragment is small (100bp).  Besides, our
institute has very strict regulation on phenol use ......SO!  By the way, is
there a specific grade/brand of agarose for retrieving DNA in this manner, clean
enough for ligation??
 
 



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