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The vector product obtained by ligation between a vector previously digested with antarctic phosphatase lacks 5' phosphate groups. T4 ligase can ligate it to an insert with complementary sticky ends which provides 2 phosphate groups. But there are still 2 nicks in the vector product! How are these normally repaired ?

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The nicks are not repaired during preparation. Instead, the vector (which is circular, despite the nicks) is transformed into the host where it is repaired by endogenous mechanisms, likely through base excision.

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  • $\begingroup$ Why was this down voted. Am I wrong? $\endgroup$ – canadianer Dec 25 '14 at 13:54
  • $\begingroup$ Yes it is possible. However the efficiency reduces. I had seen someone doing it for site-directed mutagenesis. So transforming the plasmid without sealing nicks does produce colonies but they were less than the case when the nicks were sealed. <I don't know the reason for downvote> $\endgroup$ – WYSIWYG Mar 1 '15 at 9:50

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