Uncut vector DNA-again

Sailesh Surapureddi Sailesh.Surapureddi at mcb.liu.se
Sat Apr 27 18:23:52 EST 1996


Shanthi Adimoolam <shanth at students.uiuc.edu>: writes


>Hi netters,
>In my previous posting on the uncut vector problem, i forgot to state that
>i was doing this in order to avoid vector purification from the gel,
>becasue for some reason, the ligation efficiency seems really low when i
>purify the vector form the gel that i get no transformants at all. SO i
>decided to prevent gel elution by digestion with KpnI/PstI and treatment
>with CIAP to prevent its own insert (wildtype) from ligating back, but when i
>throw in the mutant insert, hopefully that will go in becasue it still has
>its phosphate groups.
>
>I am still unable to solve the problem of uncut vector left over. Please
>help!!
>
>-shanthi


Hello Peace (Shanthi)

       KpnI is a bad cutter and it needs a lot of enzyme (NEB, advices
atleast 20units and 16hr digestion), compared to it PstI is better (though
its a lousy cutter too). So If I were you, I would allow the digestion to
go for a day in the presence of SSTK buffer with 20 units of KpnI and
5units of PstI and then gel purfiy the cut vector. Qiaex or Geneclean
should give you a good ligatable DNA. I would do the same for the insert
too (in this case your mutant one) and then ligate by sonication. This
should solve your problem. Usually one doesnt have to use CIAP etc etc for
ligations (they some time kill the ligation mix or the cells).

You are most welcome if you still have problems,
but hope you wouldnt have any.

Goodluck
Sailesh.









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