Bizzare digestion pattern

GWDGV1::KCHOWDH kchowdh at gwdgv1.dnet.gwdg.de
Fri Jun 17 07:57:48 EST 1994


Hi,
Are you quite sure about the orientation of the 600 bp plasmid?
Is it possible that this clone has the insert in the other orientation such 
that both the RI and BstEII sites are next to each other? Just a quick thought. 
Kamal Chowdhury.


	OK clone jockeys, here's an interesting set of observations for
you.  Perhaps someone out there will have an idea what they mean, and
(more importantly) a solution....
	I have two small PCR products (184 bp and 600 bp) which are blunt
ended. Both have restriction sites near the ends: BstEII and PvuI on the
small one, BstEII and XbaI on the other.  Both are cloned into the SmaI
site of pUC19, and restriction digests confirm their presence and
orientation.
	I am trying to put the products together at the common BstEII
site.  To do this I digest the small-fragment clone with EcoRI and BstEII
(which are 22 bp apart) to create a vector; I also digest the other clone
with the same enzymes (the sites being 602 bp apart).
	The small clone digests fine, either with or without
precipitation and switching of buffers between the two enzymes.  I can
even see the 22 bp fragment on agarose.
 	
	
				
				garose.
	The other clone WILL NOT DOUBLE DIGEST.  Either enzyme will
linearize the plasmid effectively; but I cannot get any visible
production of the 602 bp fragment.  I have tried changing the order of
the digests, digesting with one, ppt'ing and switching buffers between
digests, even doing one digest, gel isolating the plasmid, then doing the
second digest.  No go...so both enzymes cut, but not both.
	I even recloned the PCR product in case I just had a screwy clone
the first time around.  Same results, and I am running out of ideas (and
patience.....)
	Any theories or ideas out there?



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