ER at total.net
ER at total.net
Thu Jul 24 17:08:24 EST 1997
christoph.kruell at t-online.de (Christoph Krüll) wrote:
>Annette C. Hollmann wrote:
>> In article <33CF8404.1D91 at uva.pcmail.virginia.edu> rjl6n at virginia.edu
>> >Steven Sullivan wrote:
>> >> Has this ever happened to you?
>> >> I set up several ligations, using gel-purified double-digested DNA
>> >> vector DNA had even been dephosphorylated to ensure low background,
>> >> although this shoudl not be necessary if the double digest was
>> >> 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 .
>> >> *none* of these colonies had inserts, as assessed by PCR or miniprep.
>> >> 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
>> Also - what ratio of insert to vector are you using?
>> I was told to use a 10-fold molar excess of insert.
>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.
Can you have two or more origins of replication on a single plasmid
and have it successfully replicate in a bacterium?
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