Ligation mystery

Gail Otulakowski gotulak at sickkids.on.ca
Fri Jul 18 10:57:29 EST 1997


Steven Sullivan wrote:
> 
> Has this ever happened to you?
> 
> I set up several ligations, using gel-purified double-digested DNA (the
> vector DNA had even been dephosphorylated to ensure low background,
> although this shoudl not be necessary if the double digest was complete).
> After transformantion and growth overnight on LB/amp plates, the
> vector-only control plates were blank as expected.  The experimental
> plates had varying abounts of colonies, depending on the insert . However,
> *none* of these colonies had inserts, as assessed by PCR or miniprep.  (I
> should perhaps mention taht the vectors pCS2+ and pCS2+MT, are not
> blue/white selectable. )
> 
> I don't understand how empty plasmids can religate and grow on the
> experimental plates but not on the control.


I've had this happen when trying to subclone PCR products, but not with 
DNA excised from another vector.  I assume leftover primer or 
primer-dimers has been the problem - gives a small insert.  You can try 
double-purifying your insert DNA.

Gail Otulakowski



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