Problem with dephosphorylation

M.Albrecht M.Albrecht at em.uni-frankfurt.de
Mon Feb 12 19:51:45 EST 1996


Hi netters,
We have some problems in dephosphorylating our vector DNA (eg pUC, pQE). 
We use the alkaline phosphatase from Boehringer in combination with a 
selfmade CIP-buffer (1 mM ZnCl2, 1 mM ZnCl2, 10 mM Tris-HCl, pH 8,3) 
directly after the restriction assay. The incubation is at 37°C for about 
30 min. After this we incubate another 10 min at 75°C and then 
EtOH-precipitate the DNA before we use it in the ligation reaction. But 
after all this our vector still shows a very high rate of religation. Has 
anyone out there an idea what to do about this problem? I'll appreciate 
every useful comment. Thank you. Manuela



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