Clean-up DNA for ligation

BENNY SHOMER 9238 pc386 at ccsg.tau.ac.il
Mon Jul 19 12:22:20 EST 1993


lees at afrc.ac.uk wrote:
<Stuff Deleted>

: 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....

Yes. You need to do extraction with an equal (or more) volume of saturated 
butanol to eliminate EthBr. (Sorry, I didn't write it in the original posting).
BTW, I mention again, I'm not the original poster of this method. I'll look-up
his/her name tommorow in my archives...

  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)...

You may add carrier (glycogen, tRNA etc.), spin longer and best is - 
read the excellent summary on EtOH precipitation that appeared here, and is
in the FAQ list as well.

  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??


I don't think so. I did it with plain agarose at 1 - 2%, as well as with 
SeaKem's special brands at 4% and obtained good results (Including good 
ligations...)

Benny.

 **************************************************************
* Benny Shomer                                                 *
* Tel-Aviv University                                          *
* Sackler School of Medicine, Dept.of Embryology and Teratology*
*--------------------------------------------------------------*
* Snail:  Ramat-Aviv , Tel-Aviv  69978 ,  Israel.              *
* E-mail :  pc386 at ccsg.tau.ac.il                               *
* Tel :  972-3-640-9238     FAX :   972-3-642-2046             *
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