uncut vector DNA

corboy at utdallas.edu corboy at utdallas.edu
Tue Apr 30 14:37:22 EST 1996


George Rutherford (gruther at bilbo.bio.purdue.edu) wrote:
> Personally, I wouldn't bother to CIP a directional cloning in the first
> place on the general theory that the less unnecessary stuff you do, the
> fewer problems you get, and, in the case of CIP, if you don't kill it real
> well, you can get real problems with your ligations (they won't work
> because you CIP your insert in the ligase buffer). 

	I agree with this, since theoretically the double-cut vector
cannot re-ligate to itself.  KpnI and PstI should work together, so your
incomplete digestion may be due to inadequate enzyme or digest time, or
dirty DNA.  But regardless, you should gel-purify the cut vector before
using it in ligations or transformations.  While some may deem this step
uneccessary, think of the time it would have saved you in trouble
shooting.  With a double-cut, gel-purified vector, you should get
practically zero background, and it only takes half a day to run the gel,
cut out the bands, and get the DNA (for example with glassmilk or
agarase). 

> about anything.  Having said that, I would try to screen with colony
> hybridization. It doesn't take long and you can screen thousands at once.
> Best of luck.
> George R.

	This is way too complicated for this problem.  There is no 
foreseeable reason to screen thousands of colonies to get a ligation 
product when all you are doing is swapping restriction fragments.  Try to 
fix the original problem (high background because of incomplete 
digestion) instead of making the problem worse.  As you just said, the 
less things you do, the less places to screw up.  Think simple.  As they 
say in medical school, if you hear hoofbeats, think horse not zebra.

Michael J. Corboy
University of Texas at Dallas



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