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Enzyme Finder NEBcutter NEBuffer Chart Double Digest Finder Isoschizomers DNA Sequences and Maps REBASE

Home > Products > Protein Tools > Proteases > Proteinase K > FAQ Proteinase K FAQ See the Protein Tools FAQ also. Q1: What are the guidelines for using Proteinase K? Q2: What is the Proteinase K activity in commonly used buffers? Q1: What are the guidelines for using Proteinase K? A1: Isolation of high molecular weight DNA Chromosomal DNA that has been embedded in agarose plugs can be treated with Proteinase K to inactivate rare-cutting restriction enzymes used to digest the DNA. Proteinase K is used for this method at a concentration of 1mg/ml in a buffer containing 0.5M EDTA and 1% N-lauroylsarcosine (v/v). Incubate 24-48 hours at 37°C. Isolation of plasmid and genomic DNA Genomic or plasmid DNA can be isolated from liquid nitrogen frozen cells or cultured cells using Proteinase K. Incubate 50-100 mg of tissue or 1x108 cells in 1 ml of buffer containing 0.5% SDS (w/v) with ProteinaseK at a concentration of 1mg/ml, for 12-18 hours at 50°C. Isolation of RNA For cytoplasmic RNA isolation, centrifuge the cell lysate, remove the supernate and add 200ug/ml Proteinase K and SDS to 20%(w/v). Incubate for 30 minutes at 37°C. Total RNA can be isolated by passing the lysate through a needle fitted to a syringe prior Proteinase K treatment. Inactivation of RNases , DNases and enzymes in reactions Proteinase K is active in a wide variety of buffers (see FAQ "What is the Proteinase K activity in commonly used buffers?"). The enzyme should be used at a ratio of approximately 1:50 (w/w, proteinase K:enzyme ). Incubation is at 37°C for 30 minutes. Q2: What is the Proteinase K activity in commonly used buffers? A2: Buffer: 30 mM Tris HCl, pH 8.0 Application: Reference Relative activity (approx.): 100% Buffer: 50mM Tris-HCl, pH 8.0, 1mM CaCl2,3mM DTT, 2.0M Urea Application: Denaturation of proteins Relative activity (approx.): 70% Buffer: 100mM Tris-HCl, pH 8.0, 100mM EDTA, 250mM NaCl, 1% Sarkosyl Application: Plant tissue DNA isolation Relative activity (approx.): 120% Buffer: 10mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.5% SDS Application: Bacterial DNA isolation Relative activity (approx.): 100% Buffer: 10mM Tris HCl, pH 8.0, 50mM NaCl, 5mM EDTA, 1mM DTT, 0.5% SDS 50mM Tris-HCl, pH 8.0 Application: Denaturation of CIP Relative activity (approx.): 100% Buffer: 50mM EDTA, 5% Tween 20, 0.5% Triton-X 100, 800mM GuHCl Application: Relative activity (approx.): 300%