![]() Add 1 L 0.5 M EDTA, pH 8.0 to stop the reaction. Incubate the reaction mixture at 37 C for 1 hour. Incubate the mixture in a boiling water bath for 5-10 minutes and then chill on ice. et al., eds., John Wiley and Sons, New York, NY. Prepare the following reaction mixture: 2. (1987) In: Current Protocols in Molecular Biology, Ausubel, F.M. Ĭhoose Your Configuration: Learn more about our custom options for this product at: References The DNA-dependent DNA polymerase is provided with 10X Reaction Buffer. Add: Fluorescein-12-dUTP, DIG-dUTP or Aminoallyl-dUTP can also be used with the same protocol. Applications Random-primed DNA labeling (2-4). ![]() Research Laboratories) in a total volume of 10 pA and incubated at room temperature for 60 min. The 3'5' exonuclease activity of the enzyme is eliminated by mutations in the 3'5' exonuclease active site (1). U of the Klenow fragment of DNA polymerase I (Bethesda. It exhibits 5'3' polymerase activity, but lacks the 3'5' and 5'3' exonuclease activities of DNA Polymerase I. Klenow Fragment is used primarily in dideoxy sequencing reactions (1,2), fill-in of restriction endonuclease termini (3), and second-strand cDNA synthesis (4). Prepare the following reaction mixture: 2. Klenow Fragment, exo, is the Large Fragment of DNA Polymerase I. The 3´→5´ exonuclease activity can be used to generate blunt ends from a 3´-overhang. This protocol is for Non-radioactive Random-primed DNA Labeling with Klenow Fragment, exo. The 5´→3´ polymerase activity of Klenow Fragment can be used to fill in 5´-protruding ends with unlabeled or labeled dNTPs, to sequence single- or double-stranded DNA templates, for in vitro mutagenesis using synthetic oligonucleotides, for cDNA second-strand synthesis and to generate single-stranded DNA probes. coli DNA Polymerase I that lacks the 5´→3´ exonuclease activity of intact DNA polymerase I but retains its 5´→3´ polymerase, 3´→5´ exonuclease and strand displacement activities. ![]() DNA Polymerase I Large (Klenow) Fragment is a 68kDa C-terminal fragment of E. Klenow assembly method: seamless cloning Authors: Md David David MD Bailey Mohamed Yh Abstract Effective, ligation free, seamless cloning for overlapping double stranded DNA, using the Klenow. ![]()
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