Answer: I can help you with some information and resources about restriction digestion, a procedure used in molecular biology to prepare DNA for analysis or other processing. Restriction digestion involves using enzymes called restriction endonucleases that cleave DNA at specific sequences. There are hundreds of different restriction enzymes, allowing scientists to target a wide variety of recognition sequences. Restriction digestion is commonly used in molecular cloning techniques, such as PCR or restriction cloning. It is also used to quickly check the identity of a plasmid by diagnostic digest.
To perform a restriction digestion, you will need the following:
DNA: The DNA that you want to cut with the restriction enzymes. This can be plasmid DNA, genomic DNA, or PCR product. The amount of DNA that you cut depends on your application. A diagnostic digest typically involves ∼500 ng of DNA, while molecular cloning often requires 1 µg of DNA.
Restriction enzymes: The enzymes that will recognize and cut the specific sequences in your DNA. You can use one or more enzymes depending on your purpose. You will need to check the manufacturer’s instructions for the proper amount and storage conditions of the enzymes. You will also need to determine the best buffer that works for your enzymes, especially if you are conducting a double digest (digesting with two enzymes at the same time).
Buffer: The solution that provides the optimal conditions for the restriction enzymes to function. The buffer usually contains salt, pH stabilizers, and sometimes BSA (bovine serum albumin) to enhance enzyme activity. You will need to use the appropriate buffer for your enzyme(s) and follow the manufacturer’s instructions for the concentration.
dH2O: Distilled water to adjust the total volume of your reaction.
Gel loading dye: A solution that contains a tracking dye (such as bromophenol blue) and a density agent (such as glycerol) to help you load your DNA samples onto an agarose gel for electrophoresis.
A typical restriction digestion protocol is as follows:
In a 1.5 mL tube, combine the following components:
DNA
Restriction enzyme(s)
Buffer
BSA (if recommended by manufacturer)
dH2O up to total volume
Mix well and incubate at the appropriate temperature (usually 37°C) for 1 hour or longer depending on your enzyme(s).
Add gel loading dye to your digested DNA samples and load them onto an agarose gel along with a DNA ladder (a mixture of DNA fragments of known sizes) as a reference.
Run electrophoresis at a suitable voltage (usually 100-120 V) for an appropriate time depending on the size and resolution of your DNA fragments.
Visualize your DNA bands using a UV transilluminator or a gel documentation system. Compare the sizes and patterns of your bands with the expected results based on your plasmid map and restriction sites.