bizzare digestion problem

Sean David Moore smoore at mail.sas.upenn.edu
Fri Jun 17 09:19:54 EST 1994


Dear John (ha ha ha)


I had a similar problem with a similar experiment...I bought new
enzyme..re-preped the sample..and it worked fine.  The fact that both are
cutting just fine is strange.  is it possible that a site is lost or
changed...you are seeing non-specific linearization from one of the
enzymes..naaa..
	hmmm...
if your insert in in a MCR, try double digesting it out with two outside
of the insert.  Remember you are dealing with PCR products..and lord knows
what the sequence is.  If oyu can simply double jdigest the vector
out...then I would go back and try to continue the experiment.  Maybe, for
some reason, you particular double-D procedure is in error.


Strange/but true

Uncle Sean
X









John Brunstein (brunstei at UNIXG.UBC.CA) wrote:
: 	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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