Ligation mystery

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
>> 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.

Can you have two or more origins of replication on a single plasmid
and have it successfully replicate in a bacterium?
-ER



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