The Klenow Fill-in Kit contains Klenow fragment (125 U), dNTPs. mutagenesis using synthetic oligonucleotides, for cDNA second-strand. Or, use the kit to radioactively end label a 5 overhang with alpha -32 PdNTP. DNA Polymerase I Large (Klenow) Fragment is a 68kDa C-terminal fragment of E. Choose the Klenow Fill-In Kit to perform both partial fill-in reactions to complete fill-in of a 5 overhang to generate blunt ends, and allow direct ligation.
#Klenow fragment mutagenesis full size
The enzyme is supplied in 20 mM Tris-HCl, 1 mM DTT, 0.1 mM EDTA, 50 glycerol pH 7.5 25C. Klenow, Full Size and Exonuclease Deficient. Based on these results and the studies by others in various systems, we conclude that family A and B DNA polymerases, at least in the 3'->5' exonuclease domain, are structurally as well as evolutionarily related. Klenow Fragment (3'5' exo-) is a mesophilic DNA polymerase that displays a moderate strand displacement activity during DNA synthesis. The metal binding ligands are also essential for the strand-displacement activity of the PRD1 DNA polymerase.
Site-directed mutagenesis studies revealed that the residues important for the 3'->5' exonuclease activity, particularly metal binding ligands for the Klenow fragment, are all conserved in the PRD1 DNA polymerase as well. Although the PRD1 DNA polymerase has a smaller 3'->5' exonuclease domain, its active sites appear to be very similar to those of the Klenow fragment. In order to establish the evolutionary relationship between the family A and B DNA polymerases, we have closely compared the 3'->5' exonuclease domains between the Klenow fragment of E.coli DNA polymerase I (a family A DNA polymerase) and the bacteriophage PRD1 DNA polymerase, the smallest member of the DNA polymerase family B.
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