8/27/2023 0 Comments Shelf life of klenow fragment![]() 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 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. Unit activity was measured using a two-fold serial dilution method. 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.Add dNTPs (each at a final concentration of 33 µM).
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