New Search

Export article

Labeling of Synthetic Oligonucleotides Using the Klenow Fragment of E. coli DNA Polymerase I

Michael R. Green, Joseph Sambrook

Abstract: In this method, a short primer is hybridized to an oligonucleotide template whose sequence is the complement of the desired radiolabeled probe. The primer is then extended using the Klenow fragment to incorporate [α-32P]dNTPs in a template-directed manner. After the reaction, the template and product are separated by denaturation followed by electrophoresis through a polyacrylamide gel under denaturing conditions. With this method, it is possible to generate oligonucleotide probes that contain several radioactive atoms per molecule of oligonucleotide and to achieve specific activities as high as 2 × 1010 cpm/µg of probe. Because the end product of the reaction is dsDNA, whose strands must be separated and the labeled product isolated, this method is generally not used to prepare nonradiolabeled oligonucleotides.
Keywords: oligonucleotide / Klenow / denaturing / electrophoresis / nonradiolabeled / gel / cpm/µg / atoms

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

Share this article

Click here to see the statistics on "Cold Spring Harbor Protocols" .
References (8)
    Back to Top Top