#KLENOW FRAGMENT PDF SERIAL#

coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. DNA Polymerase I, Large (Klenow) fragment was originally derived as a proteolytic product of E.coli DNA polymerase that retains polymerase and 3’ > 5’ exonuclease activity. Note: Klenow Fragment, Exonuclease Minus, will leave a single-base 3´-overhang for a significant proportion of the DNA fragments during the fill-in reaction (8). Stop the reaction by heating the mixture for 10 minutes at 75☌. Incubate the reaction at room temperature for 10 minutes.

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. unit of Klenow Fragment per microgram of DNA. Klenow retains the polymerization fidelity of the holoenzyme without degrading 5 termini. The enzyme lacks the 5’3 exonuclease activity of intact 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. What is a klenow fragment Klenow fragment: Definition and Applications Definition: It is the large fragment of DNA polymerase I enzyme of E. Klenow Fragment is the large fragment of DNA Polymerase I that retains its 5’3 polymerase, 3’5 exonuclease and strand displacement activities. 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. Physical purity is evaluated by SDS-PAGE of concentrated and diluted enzyme solutions followed by silver-stain detection. 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. coli is cleaved by the protease enzyme subtilisin.

#KLENOW FRAGMENT PDF SERIAL#

Unit activity was measured using a two-fold serial dilution method. The Klenow fragment is a large protein fragment which is 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. Add 1 U of Klenow Fragment per microgram of DNAĢ.coli DNA Polymerase I but lacks the 53 exonuclease activity. Add dNTPs (each at a final concentration of 33 µM) The Klenow Fragment retains the DNA-dependent DNA polymerase activity of the E.

Klenow fragment pdf