Does TE buffer inhibit restriction digestion?
Use simple TE for elution, if you have any doubt on elution buffer. Generally, It should not hamper restriction digestion.23 Nov 2018
What is the purpose of the buffer in a restriction enzyme reaction?
Major function of the buffer is to maintain pH of the reaction (usually, 8.0) and provide a favorable environment for the enzyme to function.
What affects restriction enzyme digestion?
The digestion activity of restriction enzymes depends on the following factors: Temperature: Most endonucleases digest the target DNA at 37 °C with few exceptions. … Cofactors: Restriction endonucleases require certain cofactors or combination of cofactors to digest at the recognition site.
How do you stop restriction digest?
Restriction Enzyme Digest Protocol
- Add components to a clean tube in the order shown: …
- Incubate the reaction at digestion temperature (usually 37 °C) for 1 hour.
- Stop the digestion by heat inactivation (65 °C for 15 minutes) or addition of 10 mM final concentration EDTA.
Why did my restriction digest not work?
Incomplete or no digestion due to enzyme activity blocked by DNA methylation. If your enzyme is active and digests the control DNA and the reaction is set up using optimal conditions, but you still see issues with digestion, it might be because the enzyme is inhibited by methylation of the template DNA.
Which buffer is used in restriction digestion?
The recommended buffer and/or the universal Tango buffer are supplied with each enzyme. Tango buffer has been designed for double digestions of DNA with conventional restriction enzymes.
What is a digestion buffer?
Differex™ Digestion Buffer is used in differential extractions of samples that contain a mixture of epithelial and sperm cells. A digestion combination of this buffer and Proteinase K can be used to selectively lyse epithelial cells while leaving sperm cells intact.
How can restriction enzyme digestion be improved?
Addition of a DNA oligonucleotide containing the recognition sequence, or spermidine, may improve the activity of restriction enzymes that require at least two sites for optimal digestion (e.g., AarI, SfiI).