Ligation mystery

Christoph Krüll christoph.kruell at t-online.de
Sun Jul 20 14:24:22 EST 1997


Annette C. Hollmann wrote:
> 
>  In article <33CF8404.1D91 at uva.pcmail.virginia.edu> rjl6n at virginia.edu
> writes:
>  >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.
>  >--
> 
> I had this happen to me also.
> A helpful postdoc suggested that I increase the DNA concentration, and it
> worked.
> Also - what ratio of insert to vector are you using?
> I was told to use a 10-fold molar excess of insert.
> 
> Annette

I Made this experience, too. Even double digested Vectors with non
compatible ends religated. I investigated this further, the vectors
religated as head to head dimers. So, even with blue white screening
they led to false results, because this head to head dimers resulted in
white colonies, of course.



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