Ligation mystery

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
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






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