Ligation mystery

Victor Levenson levenson at uic.edu
Fri Jul 18 15:30:46 EST 1997


sullivan at gwis2.circ.gwu.edu (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.

Well, it all depends... 

When I used StuI to get blunt end of the insert (the other end sticky)
and did not kill the enzyme it carried over through agarose gel
purification and into ligation mix (I use a "squeeze extraction
technique"). As a result I got empty "vector-alone" plate and a bunch
of colonies on the other; however none of them (well, I screen 4 to 12
usually) gave me the recombinant. Instead, all plasmids contained
shortened vector. It turned out that the vector contained StuI site
that, when cut, eliminated the sticky end of the vector and this
carryover was doing just that. When I heat-killed StuI in the insert
sample and then assembled ligation mix everything worked beautifully.

Hope that helps,

Good luck,




Victor Levenson
Res Asst Prof
UIC
levenson at uic.edu




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