EFFICIENT 5'-END LABELING OF DEPHOSPHORYLATED DNA

Efficient 5'-end labeling of DNA is an important procedure in recombin ant DNA technology. Prior to labeling, it is important to inactivate a lkaline phosphatase, used in the dephosphorylation of the DNA, by usin g proteinase It. Removal of proteinase K is usually performed by extra cting twice with chloroform:isoamyl alcohol. In this report we show th at extracting the sample four times with chloroform results in more ef ficient removal of sodium dodecyl sulfate (SDS), an important constitu tent of proteinase K buffer, which allows a 25- to 40-fold increase in labeling efficiency compared with extracting twice or once with chlor oform, respectively. Unremoved SDS inhibits efficient labeling, possib ly by inhibiting the activity of the kinase. (C) 1997 Academic Press.