self religation of double-cut plasmid vectors

HARDIES at THORIN.UTHSCSA.EDU HARDIES at THORIN.UTHSCSA.EDU
Wed Jan 4 11:19:57 EST 1995


mahmoud (dbell at julian.uwo.ca) wrote:
: Hi 

: I was wondering in your hands, if you purify your double digested plasmid 
: vector (any plasmid vector), and run a self religation experiment, what sort 
: of transformation rate do you see? (how many colonies?) Of course I need to 
: know how this rate is compared to the transformation rate of your non-cut 
: plasmid control?  
: In theory it should be zero, but...?

And AL R. PLUMMER replied:

> Of course there are a lot of variables here, but in my hands there are always
> a few transformants in a restricted, non-ligase treated plasmid prep.  I
> have always assumed that these were due to incompletely digested samples.
> With ligase I generally obtain anywhere from 95-100% more 'true' transfor-
> mants than without ligase treatment...

I vaguely remember that the original EcoRI cloning paper by Carbon was without
ligase ??  So there may be some low rate at which vector and insert get in,
associate by the sticky ends, and get fixed by bacterial repair synthesis.
I'd expect/require it to be less than 1/1000 of the supercoiled control, and
I agree with Al Plummer that in practice your background is more likely
from incomplete digestion.  Since you presumably plan to either phosphatase
the clipped out piece or remove it, I am curious as to why you are interested
in this rate??

Steve Hardies, Dept. of Bioch., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu




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