Annette C. Hollmann
ah690549 at bcm.tmc.edu
Fri Jul 18 15:09:29 EST 1997
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.
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