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
More information about the Methods
mailing list