Efficient 5′ End Labeling of Dephosphorylated DNA☆

Abstract Efficient 5′-end labeling of DNA is an important procedure in recombinant DNA technology. Prior to labeling, it is important to inactivate alkaline phosphatase, used in the dephosphorylation of the DNA, by using proteinase K. Removal of proteinase K is usually performed by extracting twice with chloroform:isoamyl alcohol. In this report we show that extracting the sample four times with chloroform results in more efficient removal of sodium dodecyl sulfate (SDS), an important constitutent of proteinase K buffer, which allows a 25- to 40-fold increase in labeling efficiency compared with extracting twice or once with chloroform, respectively. Unremoved SDS inhibits efficient labeling, possibly by inhibiting the activity of the kinase.