![]() ![]() coli DNA polymerase I that catalyzes the 53 synthesis of DNA but has lost the 53. coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. The Klenow Fragment is a proteolytic product of. coli contamination is evaluated using 5 µL replicate samples of enzyme solution that are denatured and screened in a TaqMan qPCR assay for the presence of contaminating E. It exhibits 53 polymerase activity, but lacks the 35 and 53 exonuclease activities of DNA Polymerase I. Single-stranded exonuclease is determined in a 50 µL reaction containing a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37☌.ĭouble-stranded exonuclease activity is determined in a 50 µL reaction containing a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37☌.ĭouble-stranded endonuclease activity is determined in a 50 µL reaction containing 0.5 µg of plasmid DNA and 10 µL of enzyme solution incubated for 4 hours at 37☌.Į. Thermo Scientific Klenow Fragment, exo-, is the large fragment of DNA polymerase I. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the band's mass corresponding to the protein of interest in the diluted sample. Klenow 10X Buffer (M195A): The Klenow 10X Buffer supplied with this enzyme has a composition of 500mM Tris-HCl (pH 7. Size (units) M220A 150 M220C 500 Enzyme Storage Buffer: Klenow Fragment is supplied in 50mM Tris-HCl (pH 7.5), 1mM DTT, 0.1mM EDTA and 50 (v/v) glycerol. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. DNA Polymerase I Large (Klenow) Fragment: Part No. Protein concentration is determined by OD 280 absorbance. Reactions were incubated for 10 minutes at 37☌, placed on ice and analyzed using the method of Sambrook and Russel (Molecular Cloning, v3, 2001, pp. Dilutions of the enzyme were made in a 50% glycerol Klenow (3ʹ→5ʹ exo–) storage solution and added to 50 µL reactions containing calf thymus DNA, 1x Klenow Reaction Buffer, 3H-dTTP and 100 µM dNTPs. Product Overview Documents Thermo Scientific Klenow Fragment is the large fragment of DNA polymerase I. coli is enzymatically cleaved by the protease subtilisin. Unit activity was measured using a two-fold serial dilution method. The Klenow fragment is a large protein fragment produced when DNA polymerase I from E. Stop the reaction by adding EDTA (final concentration of 10mM) and heating at 75☌ for 20 minutes. Incubate the reaction mixture at 25☌ for 15 minutes.ģ. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5 termini. The enzyme lacks the 53 exonuclease activity of intact DNA polymerase I. Add 1 U of Klenow Fragment per microgram of DNAĢ. Klenow Fragment is the large fragment of DNA Polymerase I that retains its 53 polymerase, 35 exonuclease and strand displacement activities.Add dNTPs (each at a final concentration of 33 µM). ![]()
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