Efficient 5' end labeling of dephosphorylated DNA.
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 constituent 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.
Kurien
B T
BT
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190, USA.
Scofield
R H
RH
Broyles
R H
RH
D017122:Bacteriophage T4 / Q000737:chemistry / Q000235:genetics; D002725:Chloroform; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D019286:Endopeptidase K / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods*; D010766:Phosphorylation; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D012967:Sodium Dodecyl Sulfate; D002725:Chloroform; D004247:DNA; D019286:Endopeptidase K