Heop - cloning into dephosphorylated plasmid vector

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Sat May 27 15:56:53 EST 1995


Rafael Maldonado writes:

> I normally make my ligations without dephosphorilation and 
they work well. I got 30% of colonies with insert, and when you have some 
kind of screening/selection, who cares about dephos? Or, in 10 minipreps 
you can have three insert clones.

> I only dephos when I need extremely efficient ligations (>90%), i.e., 
libraries, very long inserts, etc.

Yes, but to do this you're probably using insert:vector ratios of 10:1
or more.  This will give multiple inserts, particularly when cloning
from a mixture of potential insert fragments.  So I'd recommend
dephosphorylation and V:I ratios around 1:1 for any experiment where
multiple insertion would confuse the result.  Remember, molar ratios,
not ug ratios.

Dephos. also helps a lot if you're constrained to work with an inadequate
amount of insert, or if only a small portion of the insert is clonable 
for some reason (a problematic end repair, or damage to ends by
extraneous activities in the R. enz., etc.).

There's not too much of a downside to dephos.  You do have to remove the
phosphatase activity, but this isn't that hard once you are aware of the
problem.  It usually only knocks down the background 10-100x.  And it
also enriches for abberant clones, particularly those involving deletion
of portions of the vector.  So you still have to characterize your clones;
you can't just assume that every colony is a simple insert.

Going back to the question that kicked off this thread [a lot of plain
vector clones coming through after CIP treatment]: The most common
cause of this is when the vector isn't cut to completion in the first
place.  CCC vector transforms 10-100x better than ligation product; so
even a few % left uncut can swamp the transformation.

Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu



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