A couple of questions

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.

Why is my restriction digestion not working?

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.

What is needed for restriction digestion?

The components of a typical restriction digestion reaction include the DNA template, the restriction enzyme of choice, a buffer and sometimes BSA protein. The reaction is incubated at a specific temperature required for optimal activity of the restriction enzyme and terminated by heat.

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).

Why would restriction enzymes not work?

There can be several different reasons why your restriction enzyme does not cut the DNA as reviewed in this video. The preparation of DNA to be cleaved should be free of contaminants such as phenol, chloroform, alcohol, EDTA, detergents, or excessive salts, all of which can interfere with restriction enzyme activity.

How do you know if your restriction digestion was successful?

If the digested product would be visible at a lower coordinate on the gel, it would have made things easy. You can amplify your digested fragment with primer beginning in the flankers region and with only 3-4 bp in the intern 8680 bp region. If you do not get PCR fradments, was the digestion successfully.

What happens if you add too much restriction enzyme?

Incomplete digestion is a frequently encountered issue when using restriction endonucleases. Incomplete digestion may occur when too much or too little enzyme is used. The presence of contaminants in the DNA sample can inhibit the enzymes, also resulting in incomplete digestion.

What is a restriction enzyme do?

A restriction enzyme is an enzyme isolated from bacteria that cuts DNA molecules at specific sequences. The isolation of these enzymes was critical to the development of recombinant DNA (rDNA) technology and genetic engineering.