NEB Alkaline phosphatase contain Nuclease?

Tracy Aquilla aquilla at salus.med.uvm.edu
Tue Jul 9 13:37:50 EST 1996


In Article <960708114237.c3273a at chemvx.tamu.edu>, KHUANG at CHEMVX.TAMU.EDU wrote:
>Hi, Netters,
>We met a problem about using NEB alkaline phosphatase. 
>pUC18, pBS, pGEM were cut by single enzyme (Sal1, BamH1, EcoR1) and treated 
>with NEB alkaline phosphatase according to the protocol they described, then 
>phenol extract, ethanol ppt, and then did ligation. But we found many white 
>colonies (5-10%) appeared for the contron (no insert ligation). When the 
>digested plasmid were not treated with alkaline phosphatase, only a few
>white colonies appeared.
>Does anybody out there mmeet the same problem? How to resolve this problem?

Use a different phosphatase. I like shrimp AP because it doesn't tend to
nibble like CIP does and it can be heat-inactivated at 65C.



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