Most popular

What does Proteinase K buffer do?

What does Proteinase K buffer do?

Proteinase K is used during DNA extraction to digest many contaminating proteins present. It also degrades nucleases that may be present in DNA extraction and protects the nucleic acids from nuclease attack.

How do you resuspend Proteinase K?

(20 mg/ml) Purchase as a lyophilized powder and dissolve at a concentration of 20 mg/ml in sterile 50 mM Tris (pH 8.0), 1.5 mM calcium acetate. Divide the stock solution into small aliquots and store at -20°C. Each aliquot can be thawed and refrozen several times but should then be discarded.

How do you make Proteinase K buffer?

To ensure maximum Proteinase K activity, use water that has been treated with 0.1% DEPC (DEPC, GoldBio Catalog # D-340 [CAS 1609-47-8, mw. = 162.14 g/mol]) to make the buffer. Add 1 ml of DEPC to 1 L of H2O, shake vigorously, and let incubate for 12 hours at 37°C to inactivate any RNases.

What temperature should Proteinase K be stored at?

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.

What happens if you use 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).

Why do we denature proteinase K?

First of all, you want the RNase added because it would break down contaminating RNA during your DNA isolation. And you want to use proteinase K because it will break down damaging proteins, DNases and RNases. The answer to this question is really rooted in timing and optimization.

Can you vortex proteinase K?

Always vortex the Proteinase K before use. RNase A (not provided) can be used if RNA-free DNA is required. Use RNase A at the indicated optional step in the protocol.

What is the difference between proteinase K and pronase?

Proteinase K is a proteolytic enzyme (a serine protease) that is purified from the mold Tritirachium album. Pronase (Streptomyces griseus) can be used in place of proteinase K. In some cases it may be necessary to perform a pronase self-digestion to eliminate contaminating RNase and DNase activity.

What happens if you add too much proteinase K?

Why is proteinase K used in RNA extraction?

Proteinase K is critical because it digests proteins eliminating contamination from nucleic acid preparations, in addition to inactivating the nucleases that could degrade DNA or RNA during purification.

Is proteinase K needed for RNA extraction?

What does proteinase K Do RNA extraction?

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.

When is proteinase K active in a Neb buffer?

Proteinase K is active in a wide range of buffers including all NEB specific restriction endonuclease buffers. It is highly active between pH 7.5 and 12.0 and temperatures between 20 and 60°C (1,2).

What is the concentration of Longlife proteinase K?

Proteinase K, Lyophilized. The dry, lyophilized powder is highly soluble (>50mg/ml); however, stock solutions are normally 20mg/ml. The proteinase K is completely RNase and DNase free and has a specific activity of >30 units/mg at 37°C. The recommended storage buffer is 20mM Tris-HCl (pH7.4), 1mM CaCl2, 50% glycerol.

How long is proteinase K stable in freeze thaw cycle?

Proteinase K is stable for at least 2 years at –20°C. No loss of activity is observed after 10 freeze-thaw cycles. Reaction Conditions. Proteinase K is active in a wide range of buffers including all NEB specific restriction endonuclease buffers.

What are the safety data sheets for proteinase K?

Safety Data Sheets (SDSs) are available from Product description. Proteinase K is a nonspecific serine protease that is active in the presence of SDS or urea and over a wide range of pH, salt concentrations, and temperatures.