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ligation of two DNA fragments - (Mar/21/2006 )

Hi everyone
If I want to ligate two DNA fragments which are already cloned in two vectors (TA). These fragments can be resricted from the vector by using enzymes cla1 , Bam H1 and Bam H1 and Nhe1.That means they have one end sticky and so can be ligated from one end but how can we avoid self ligation from the other end. Because self ligation will be from Bam H1 end also but if we somehow restrict self ligatin from the other end then there will be only two chances for self ligation and the third chance will be for the desired ligation of two fragments.


You can do this by dephosphorylation (alkaline phosphatase treatment, SAP or C(I)AP). With this you remove the 5'-terminal phosphate from the DNA and then it needs the phosphate of your other fragment to ligate, so it can not self-ligate anymore.