How do you reconstitute Proteinase K?
Dissolve the Proteinase K: • Dissolve in 10 mM Tris HCl, pH 7.5, 20 mM calcium chloride, and 50% glycerol, then store at –20°C. Dissolve in 50 mM Tris HCl (pH 8.0), 1–5 mM calcium acetate, then store at 2°C–8°C. Storage at –20°C in the absence of glycerol can lead to precipitation of the Proteinase K.
How long is Proteinase K good for?
QIAGEN Proteinase K is stable for up to 1 year after delivery when stored at room temperature. To prolong the shelf-life of Proteinase K, storage at 2–8°C is recommended.
Can I dilute Proteinase K in water?
As a general rule, proteinase K is stable and very active in buffers that con- tain denaturing reagents such as urea, sodium dodecyl sulfate (SDS), and guanidinium salts. Proteinase K is soluble at least up to 20 mg/ml in double-distilled water.
Why do you need to inactivate Proteinase K?
For example, in the nucleic acid extraction protocol, proteinase K is added to cell lysate and then an incubation period follows to ensure a complete digestion. To prevent potential digestion of your samples, proteinase K is inactivated after incubation. The common temperature for inactivation is 95°C.
What is proteinase K do?
Proteinase K is used for the destruction of proteins in cell lysates (tissue, cell culture cells) and for the release of nucleic acids, since it very effectively inactivates DNases and RNases.
Can you add too much proteinase K?
Proteinase K is a protein and it will constitute a trivial amount of the protein in the extract and will be removed in the Ph-CHCl3 extraction. The SDS might hang around if you use too much, especially if you ethanol precipitate (though I suspect SDS is quite ethanol-soluble).
How long does it take for proteinase K to work?
Proteinase K is active in a wide variety of 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….WHAT IS PROTEINASE K?
|Buffer (pH 8.0, 50°C, 1.25 µg/ml protease K, 15 min incubation)||Proteinase K activity (%)|
|30 mM Tris·Cl||100|
What does proteinase K break down?
Proteinase K is able to digest hair (keratin), hence, the name “Proteinase K”. The predominant site of cleavage is the peptide bond adjacent to the carboxyl group of aliphatic and aromatic amino acids with blocked alpha amino groups. It is commonly used for its broad specificity.
Why do we need to inactivate the nucleases during the DNA isolation process?
Proteinase K is commonly used in molecular biology to digest protein and remove contamination from preparations of nucleic acid. Addition of proteinase K to nucleic acid preparations rapidly inactivates nucleases that might otherwise degrade the DNA or RNA during purification.
Is proteinase K SDS resistant?
Proteinase K is itself a protein; however, it is highly resistant to denaturation by heat, chaotropic salts, and detergents (ie. SDS, sarkosyl) and will continue to function in their presence provided the temperature/concentration do not cross a fairly high threshold (up to 65°C).
What happens if you use too much proteinase K?
What is recombinant proteinase K?
Recombinant Proteinase K is a nonspecific serine protease that is useful for general digestion of proteins. For Research Use Only. Not for use in diagnostic procedures.
What is the role of Proteinase K in hybridization?
Proteinase K remains active: Removal of endogenous nucleases during the preparation of DNA and RNA; preparation of tissue sections for in situ hybridization. Endodeoxyribonuclease and exodeoxyribonuclease assays; liquid form tested for absence of RNase activity.
What are the recommended conditions for proteinase K treatment?
How do you recommend storing proteinase K? We recommend storing it at 4°C. What are the recommended conditions for proteinase K treatment when isolating RNA or DNA samples? Concentration: Generally proteinase K is used in the concentration range of 50 to 500 µg/mL at 65 degrees C in the presence of SDS (0.5-1%).
What is the concentration of Proteinase K in SDS?
Concentration: Generally proteinase K is used in the concentration range of 50 to 500 µg/mL at 65 degrees C in the presence of SDS (0.5-1%). pH: Proteinase K is stable over a wide pH range (4.0 to 12.5), with optimal activity at pH 6.5 to 9.5. It is most stable at pH 8.