bizzare digestion problem

Winoto lab lab_winoto at maillink.berkeley.edu.
Tue Jun 28 18:45:37 EST 1994


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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