Question

How do you set up a restriction digest? What are the controls? What is the purpose of controls? What are the components and what is the purpose of each component?

Answer 1

Solution -

Restriction enzymes or endonucleases are enzymes that recognize short, specific  DNA sequences. They cleave double-stranded DNA at specific sites within or adjacent to their recognition sequences.
Each restriction enzyme has specific requirements for optimal activity.

Experimental controls are necessary to identify, understand and explain problems or inconsistencies in results. The
following controls are commonly used in parallel with RE digests:

• uncut experimental DNA
• digestion of a commercially supplied control DNA
• no-enzyme “mock” digestion.

Restriction digestion is usually used to prepare a DNA fragment for subsequence molecular cloning, as the procedure allows fragments of DNA to be pieced together like building blocks via ligation.

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.