bizzare digestion problem-solved?

John Brunstein brunstei at UNIXG.UBC.CA
Wed Jun 29 13:13:15 EST 1994


	Well, thanks for all the replies I received.  Most people seemed 
to think that my problem stemmed from orientation (or in an intriguing 
twist, use of pUC18 instead of pUC19).  However, as I tried several 
diagnostic digests and all pinted to the clone being the correct 
orientation and being in pUC19, I do not think orientation is the problem.
	I should perhaps mention that the clone is of a section of a 
ssDNA virus.  This gave me suspicions that perhaps upon cutting with one 
enzyme a secondary structure was forming and blocking the other cut 
site.  One person who replied (G.Kalmar) suggested this, and recommended 
trying a PvuII digest...it should cut twice, once on each side of the 
polylinker.  This might (or might not) influence the structure and allow 
me to get my fragment.
	Well, off I went with PvuII in hand.....and lo and behold, 
although both sites cut, the fragment released runs at ~300 bp when I 
KNOW it's ~900.  I have taken this as convincing evidence of secondary 
structure and trashed the clone.  Fortunately I have another clone which 
shares much of the same region, can be used for the reconstruction, and 
is well-behaved.
	The moral of the story?  Beware when dealing with natively 
single-stranded  organisms!  I can't see (or rather my software can't 
see) any structural motifs in the region of grief, but there you have 
it... 
	Thanks again to everyone for all the suggestions!



On 28 Jun 1994, Winoto lab wrote:

> In article <Pine.3.89.9406161131.B14868-0100000 at unixg.ubc.ca>
> brunstei at UNIXG.UBC.CA (John Brunstein) writes:
> 
> >         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?
> 
> 
> Is this a contest question?  If not it should be 'cause its a good one.
>  Well heres my entry:
> 
> You have inadvertantly cloned your pcr product into pUC18 which has the
> polylinker in the reverse orientation from pUC19.  Otherwise the
> plasmids are identical.  So if you "confirmed the orientation" by using
> enzymes which cut internal to the insert but outside of the polylinker
> you would be fooled into thinking that you had the right orientation. 
> In reality, however the EcoRI site is on the opposite side that you'd
> expect.  Hence, your internal BstEII site is actually right next to
> your external EcoRI site.  This would explain why both enzymes will cut
> but do not liberate your fragment.  How'd I do?
> 
> Pendragon
> 
> 



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