Self religation of double-cut plasmid vectors

Lee H. Remington lremington at popmail.ucsd.edu
Wed Jan 4 14:35:06 EST 1995


In article <dbell.2.00421AE8 at julian.uwo.ca>, dbell at julian.uwo.ca (mahmoud)
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...?
> 
> 
   Maybe I misunderstood something but, in theory the plasmid can religate
to itself and give dimers, quadramers, etc....  If I do not treat with
alkaline phosphatase, I see what I presume to be multimers (this is after
subtracting out for background i.e. colonies from non ligase treated
double digests.)
   After ten years of cloning work, I am thoroughly convinced that gel
purification is a waste of time; gel purified fragments are almost always
contaminated with fragments from other parts of the gel (presumable some
DNA migrates on top of the gel and somehow contaminates the band of
interest). However, the real reason not to gel purify is that a major drop
in transformation efficiency occurs, either due to agarose gel
contamination or nicking of DNA on trans-illuminators. 
   The best advice I can give is try to do all DNA manipulations in a
microcentrifuge tube, use alkaline phosphatase instead of gel purifying
the plasmid and then screen the appropriate number of colonies to isolate
the desired construct.



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