Hello All:

Les Willis WILLISL at EM.AGR.CA
Thu Feb 15 12:59:32 EST 1996


Hello All:

     Thanks to everyone who responded to my original note on a deletion problem I was 
having. A suggestion by Joe Chou to simply re-ligate from a digest without get purification
solved the problem. I've produced all my deletions (in dH5) without any difficulty. Thanks
again.  Les.

>>> Joe Chou <jchou at cgl.ucsf.edu> 02/09/96 05:51pm >>>


>When I was working with a 40 kb cosmid containing genomic C. elegans
>DNA, I produced good deletions very similarly to the way you have been trying, also into DH5a.

>However, on the assumption that smaller constructs would transform far more efficiently, and that unimolecular
>ligations would be far more efficient that bimoleculars (i.e., I also diluted down the single enzyme restriction digest
>significantly), I cut down the manipulations to:

>  1) digest the cosmid
>  2) heat kill the enzyme
>  3) dilute down the digest, add 10x ligation buffer and ligase
>  4) transform

>I worry that during the gel purification, you are having problems with both recovery and shearing.

>I had to make MANY deletions in this fashion, and it always worked, so you might want to try being sloppy, like I
>was :)

>Joe jchou at socrates.ucsf.edu

>--
>- Joe Chou  (jchou at socrates.ucsf.edu)
>  http://devbio-mac1.ucsf.edu
>  PGP public key available by finger or at WWW page
>  PGP FP [004C 5A68 CC2F DA20  3999 3355 0E8D 7B3F]






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