The acceptance criteria for this test requires that the aggregate mass of contaminant bands in the concentrated sample do not exceed the mass of the protein of interest band in the dilute sample, confirming greater than 99% purity of the concentrated sample.Ī 50 µL reaction containing 10,000 cpm of a radiolabeled single-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37☌ resulted in greater than 50% release of TCA-soluble counts.Ī 50 µL reaction containing 5,000 cpm of a radiolabeled double-stranded DNA substrate and 10 µL of enzyme solution incubated for 4 hours at 37☌ resulted in greater than 50% release of TCA-soluble counts.Ī 50 µL reaction containing 0.5 µg of pBR322 DNA and 10 µL of enzyme solution incubated for 4 hours at 37☌ resulted in no visually discernible conversion to nicked circular DNA as determined by agarose gel electrophoresis. Following electrophoresis, the gel was stained and the samples compared to determine physical purity. The observed average measurement of 3 replicate samples was converted to mg/mL using an extinction coefficient of 55,330 and molecular weight of 68,202 Daltons.Ģ.0 µL of concentrated enzyme solution was loaded on a denaturing 4-20% Tris-Glycine SDS-PAGE gel flanked by a broad-range MW marker and 2.0 µL of a 1:100 dilution of the sample. Protein Concentration (OD 280) MeasurementĪ 2.0 µL sample of enzyme was analyzed at OD 280 using a Nanodrop ND-1000 spectrophotometer standardized using a 2.0 mg/ml BSA sample (Pierce Cat #23209) and blanked with product storage solution. Reactions were incubated 10 minutes at 37☌, plunged on ice, and analyzed using the method of Sambrook and Russel ( Molecular Cloning, v3, 2001, pp. Dilutions of enzyme were made in a 50% glycerol Klenow (3′-5′ exo-) storage solution and added to 50 µL reactions containing Calf Thymus DNA, 1X Klenow Reaction Buffer, 3H-dTTP and 100 µM dNTPs.
#Klenow fragment molecular weight serial
VUnit activity was measured using a 2-fold serial dilution method. PRODUCT SPECIFICATION* Storage Temperature coli strain carrying the Klenow Fragment gene.ġ unit is defined as the amount of polymerase required to convert 10 nmol of dNTPs into acid insoluble material in 30 minutes at 37☌. The protein is expressed as a truncated product of the E.coli PolA gene.Ī recombinant E. The enzyme exhibits DNA synthesis and proofreading (3′→5′) nuclease activities, and, in the absence of the holoenzyme’s (5′→3′) nuclease domain, displays a moderate strand displacement activity during DNA synthesis. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.Klenow Fragment is a mesophilic DNA polymerase derived from the E.coli Polymerase I DNA-dependent repair enzyme. Removal of 3´ overhangs to form blunt ends.
Fill-in of 5´ overhangs to form blunt ends.DNA sequencing by the Sanger dideoxy method.The other small fragment has 5'→ 3' exonuclease activity, but lacks 5'→ 3' polymerase activity and 3'→ 5' exonuclease activity of klenow fragment. It has 3'→ 5' exonuclease activity for proof reading activity This klenow fragment lacks 5'→ 3' exonuclease activity. On the left side, upon treatment with protease, two fragments are formed, a large fragment called the klenow fragments and a small fragment with 5'→ 3' exonuclease activity. On the right side is the DNA Polymerase I before treating with protease. Refer this figure for clear understanding.
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