What is the optimal temperature for proteinase K?
between 20 and 60°C
Proteinase K is active in a wide range of temperatures and buffers with optimal activity between 20 and 60°C and a pH between 7.5 and 12.0 (1, 2). Activity is stimulated when up to 2% SDS or up to 4 M urea are included in the reaction (3).
Why is heat inactivation of proteinase K necessary?
It is ideal for these applications because proteinase K is able to break down proteins and inactivate DNases and RNases that would otherwise degrade a desired sample of DNA or RNA.
What is the concentration of proteinase K?
50 to 500 µg/mL
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%). Temperature optimum: 65 degrees C; 12X more active at 65 degrees C than at 25 degrees C.
Why is it necessary to add RNase a before proteinase K treatment?
First of all, you want RNase to be 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!
How is proteinase K used in DNA extraction?
Proteinase K DNA extraction protocol
- Take 2 ml of the blood sample and add 10 to 20μL of TE buffer to the sample.
- Centrifuge the sample at 2500 rpm for 20 minutes.
- Discard the supernatant and add 10 to 15μL of TE buffer to the pallet and mix it gently.
- Centrifuge the sample at 2500 rpm for 15 minutes.
What is the function of proteinase K?
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.
How do you dissolve 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.
What is proteinase K used for in DNA extraction?
Applications. 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.
What is the purpose of using proteinase K in DNA extraction?
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.
What is K protocol?
Proteinase K Product Information It cleaves peptide bonds adjacent to the carboxylic group of aliphatic and aromatic amino acids and is useful for general digestion of protein in biological samples. It has been purified of RNase and DNase activities. A typical working concentration for Proteinase K is 50–100µg/ml.
What buffer should I use for proteinase K?
Proteinase K is active in a wide range of buffers including all Restriction Enzyme NEBuffers, Q5 Reaction Buffer, One Taq Standard Reaction Buffer, Standard Taq Reaction Buffer and RNAPol Reaction Buffer.
What does proteinase K do to the cell?
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 . The enzyme’s activity towards native proteins is stimulated by denaturants such as SDS. A.
What are some functions of Proteinase K?
Proteinase K is a serine protease that is used to digest proteins and remove contamination from nucleic acid preparations. In molecular biology research, adding Proteinase K to nucleic acid preparations inactivates nucleases that could degrade DNA or RNA during isolation and purification applications.
What is proteinase K’s main role in DNA extraction?
Proteinase K is used in DNA extraction to digest the tissue/cell membranes, etc. If the tissue and/or cells aren’t digested all the way, it will greatly decrease your yield (it sounds like yours weren’t digested at all which is probably why you didn’t get any bands).