Team:Waterloo/Notebook/Protocols/DNA Digestion


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Digestion of Plasmid DNA

Before you start
- Know what is the total digestion volume mix. From that you can determine the enzyme concentration which will have to be at or below 10%. You can also calculate the buffer volume based on the concentration of the buffer needed

MQ water (Vtotal - Vbuffer - VDNA - Venzymes)
Buffer (depends on required concentration: For 10x buffer, add 1/10 Vtotal
DNA (1 µL)
Enzymes (0.5 µL each/digest)
Microfuge tube (1/digest)

1. In one tube, mix the above materials in the given order.
- Keep the enzymes cold as much as possible. Do not leave them out for longer than necessary.
2. Vortex briefly.
3. Centrifuge 2-3 seconds.
4. Digest 1 hour at 37ºC.