Ligation mystery

R. John Lye rjl6n at
Fri Jul 18 09:56:04 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.

Yeah, this just happened to me, too - exactly as you
described (except that I didn't de-phosphorylate, and
I'm using a different vector).  I'm still trying to sort 
it out, and am trying to figure out what went wrong.  
Quite frustrating though.
John Lye

e-mail to: rjl6n at Virginia.EDU

More information about the Methods mailing list