et al., eds., John Wiley and Sons, New York, NY. (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. The 3´→5´ exonuclease activity can be used to generate blunt ends from a 3´-overhang. Protocol for blunting ends by 3' overhang removal and fill-in of 3' recessed (5' overhang) ends using DNA Polymerase I, Large (Klenow) Fragment (M0210) Protocols.io also provides an interactive version of this protocol where you can discover and share optimizations with the research community. 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. Site-directed mutagenesis of the large fragment of DNA polymerase I (Klenow fragment) yielded two mutant proteins lacking 3,5-exonuclease activity but. It retains polymerase activity, but lacks both 5’ > 3’ and 3' > 5’ exonuclease activity. 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. Klenow Fragment (3'5' exo-) DNA Pol I, Large (Klenow) fragment was originally derived as a proteolytic product of E. DNA Polymerase I Large (Klenow) Fragment is a 68kDa C-terminal fragment of E.
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