Deletion dilemma

Joe Chou jchou at cgl.ucsf.edu
Fri Feb 9 20:51:34 EST 1996


WILLIS at BCRSSU.AGR.CA writes:

>	I've recently been attempting to produce deletions in a 
>large (19kb) plasmid with very little success. Clones I isolate
>are typically very small (approx. 3kb). The original construct 
>is stable, although I encountered similar problems (but milder)
>when I originally constructed it. The insert is a 16kb mouse 
>mt genome in the vector pSP64 (amp resistance) with a T7 promotor
>cassette. 
>	To produce deletions I digest with a single enzyme
>(I've tried several), gel purify the band (typically 10-16 kb
>including the vector), perform a room temp. ligation and transform
>chemically competent dh5alpha. Plating results in a very large
>number of tranformants but all without the full length insert. I 
>observe a variety of different colony morphologies when I plate. 
>Previously I used this to my advantage, as the full length construct
>formed a very small colony. Unfortunately (with the deletions)
>this is no longer the case. I've selected dozens of colonies to 
>date and they are all severely deleted. 

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

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- Joe Chou  (jchou at socrates.ucsf.edu)
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