bizzare digestion problem

John Brunstein brunstei at UNIXG.UBC.CA
Thu Jun 16 14:09:10 EST 1994


	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?




More information about the Methods mailing list