How do I perform a restriction digest on total DNA isolation from mammalian cells?
Isolating whole DNA from mammalian cells and performing a restriction digest is a multi-step process. It is important to note that the specific experimental design and laboratory methods may need minor adjustments to this general technique.
Here’s a high-level framework for thinking about it:
Materials
Isolated Genomic DNA
Inhibitory Enzymes
buffer for reactions
“De-ionized” water
Sterile pipette tips
A set of six microcentrifuge tubes
microcentrifuge
Equipment for performing an agarose gel electrophoresis.
A hot water bath or a heat block
Procedure
1. First, you’ll need to isolate total DNA from mammalian cells using either a phenol-chloroform extraction or a DNA isolation kit purchased from a store.
2. After DNA purification, use a spectrophotometer or fluorometer to determine the DNA concentration.
3 Get the response for restricted digestion going. For this, mix the following: There should be one to five milligrams of DNA, one to five units of restriction enzyme per milligram of DNA (as suggested by the manufacturer of the restriction enzyme), one-tenth of a liter of 1x restriction buffer (usually supplied by the manufacturer of the restriction enzyme), and enough deionized water to make the total volume to the best working volume (20 to 50 ul).
4 Allow the reaction mixture to incubate at the temperature specified by the restriction enzyme’s manufacturer (typically 37°C). Depending on the restriction enzyme and the methods being followed in the lab, the incubation duration might be anywhere from 1 to 4 hours.
5. Heat-inactivate the enzymes at 65–80 degrees Celsius for 20 minutes after incubation (depending on the specific enzyme used).
Apply gel electrophoresis to your restriction digest for analysis.
Always refer to the manufacturer’s instructions for the restriction enzymes you use to determine the optimal reaction conditions (concentration, temperature, and incubation time). Avoid contamination at all costs by strictly adhering to aseptic procedures.
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