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KLENOW FRAGMENT MOLECULAR WEIGHT SERIAL

Please click the arrow on the right to expand the citation list. Incubation of 10 units of Klenow with supercoiled plasmid DNA produced no nicked molecules after 20 hours at 37 ☌ as determined by agarose gel electrophoresis analysis. The enzyme is greater than 98 % pure as indicated by SDS-polyacrylamide gel electrophoresis and contains no detected endonuclease activity. Dissolve 0.1 - 4 μg of digested DNA in 1x Reaction Buffer supplemented with 40 μM each dNTPĥ00 mM Tris-HCl pH 7.6 at 25 ☌, 50 mM MgCl 2 and 10 mM DTT.Įxcessive amounts of enzyme or longer reaction times may result in recessed ends due to the 3'→5' exonuclease activity of the enzyme.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.

Klenow Fragment is the large fragment of DNA Polymerase I that retains its 5'→3' polymerase, 3'→5' exonuclease and strand displacement activities. Removal of 3' overhangs to form blunt ends.Fill-in of 5' overhangs to form blunt ends.Unit Definition: One unit is defined as the amount of enzyme required to convert 10 nmoles of dNTPs to an acid insoluble form in 30 minutes at 37 ☌.įorm: liquid (Supplied in 100 mM KPO 4 pH 6.5, 1 mM DTT and 50 % glycerol) RNA Labeling Selector (Kits & Nucleotides).DNA Labeling Selector (Kits & Nucleotides).Privacy Policy for Jena Bioscience's Facebook Page.Poly (A) carrier RNA-based RNA purification.3'-End RNA Labeling (T4 RNA Ligase 1-based).Random RNA Labeling ( in vitro Transcription-based).Click Chemistry-based RNA/cRNA Labeling.Click Chemistry-based DNA/cDNA Labeling.Quantifoil® Holey Carbon Films with 2 nm Continuous Carbon.Mercurated and Selenium-containing Nucleotides.JBS Tungsten Cluster Derivatization Kits.Cryo and Room Temperature Crystallography.in Posttranslational Modification Analysis Immobilized Nucleotides for Affinity Chromatography.

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coli genomic DNA using oligonucleotide primers corresponding to the 16S rRNA locus. 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. 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. Purity is assessed by comparing the aggregate mass of contaminant bands in the concentrated sample to the mass of the band 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 10 minutes at 37☌, placed on ice, and analyzed using the method of Sambrook and Russell ( Molecular Cloning, v3, 2001, pp. Dilutions of enzyme were made in a glycerol (50%) containing Klenow (3ʹ→5ʹ exo–) storage solution and added to 50 µl reactions containing calf thymus DNA, 1x Blue Buffer, 3H-dTTP and 100 µM dNTPs.

KLENOW FRAGMENT MOLECULAR WEIGHT SERIAL

Unit activity was measured using a twofold serial dilution method.