What are the hazards of under or over digestion with a restriction enzyme by either the amounts of reagents used or time and temperature a reaction is incubated?
The hazards of under or over digestion with a restriction enzyme can occur due to several variables, including the amounts of reagents used, as well as the time and temperature of the reaction incubation. Let's break it down:
1. Under digestion: This happens when insufficient amounts of restriction enzyme are used or when the reaction is not incubated for enough time. Under digestion can lead to incomplete cleavage of the DNA sample, resulting in incomplete digestion and the presence of cutting-resistant DNA fragments. This can create problems in downstream applications like DNA ligation or cloning, as the desired fragments may not be present in the expected ratio.
To avoid under digestion:
- Ensure the correct amount of restriction enzyme is added according to the manufacturer's instructions.
- Incubate the reaction for the recommended duration, which is typically several hours or overnight.
- Follow the appropriate temperature conditions specified by the manufacturer.
2. Over digestion: This occurs when an excessive amount of restriction enzyme is added to the reaction or when the reaction is incubated for too long. Over digestion can lead to excessive cleavage of the DNA sample, resulting in completely digested fragments or even degraded DNA. This can also cause problems in downstream applications, as the desired fragments may be too short or may not be present at all.
To avoid over digestion:
- Use the recommended amount of restriction enzyme as specified by the manufacturer.
- Incubate the reaction for the recommended duration, avoiding prolonged incubation.
- Ensure the reaction is performed at the temperature recommended by the manufacturer.
It is important to carefully follow the instructions provided by the manufacturer of the specific restriction enzyme being used. These instructions typically include the optimal amount of enzyme, recommended incubation time, and temperature specific to the enzyme to achieve optimal digestion. Adhering to these guidelines will help to minimize the hazards of under or over digestion when working with restriction enzymes.