Plasmid size vs supercoiling

Peter M. Muriana muriana at aclcb.purdue.edu
Wed Jul 21 20:23:15 EST 1993


In article <44814.jdboer at sol.uvic.ca>, jdboer at SOL.UVIC.CA ("Johan de Boer") writes:
>Hi,
>When we run the (uncut) plasmid DNA from
>minipreps on an ethidiumbromide/agarose gel the major band runs at a
>certain position, but one clone runs much slower (at approximately 2.5
>and 5 kb linear markers respectively). The faster ones turn out to be
>just the vector, when we linearize the DNA with an enzyme that cuts
>one of the cloning junctions. The slow running DNA would look promising,
>however it looks exactly the same as the others after linearizing it.
>Is it possible for a plasmid from two colonies to have such different
>supercoiling patterns?
>Any ideas would really be appreciated
>
>Johan de Boer

Johan,

Think about what your saying: results from an attempted cloning experiment 
are a) clones that are vector only and run ~2.5 kb (uncut), and
    b) **tentative** clones that are running about ~5.0 kb (uncut), and
    c) when both are cut with a Res. Enz., they are appearantly the same.

Conclusion(s): i) you've got a vector:vector ligation, linearizing with
		  the R.E. used for cloning gives two, same-sized products
		  (likely reason).
	      ii) you've cloned an insert that's the same size as the 
		  vector (less likely).

	      iii) you can test hypothesis (i) by seeing if cutting at 
			other unique vector sites keeps giving you 
			one (head-to-tail ligation) or two (head-to-head 
			ligation) digestion products.
		  
		 		
Regards, Peter Muriana



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