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Team:Brown/Notebook Protocols/redigest
From 2009.igem.org
DNA digestion protocol & hints
Overview: Although it is pretty standard to digest DNA with restriction enzymes, here is a standardized protocol and some hints…
Materials:
• DNA sample in water or TE buffer
• 10x digestion buffer
• restriction enzyme
• DNA loading buffer
• Agarose gel 0.8% (or different depending on expected band sizes)
Procedure:
1. Pipet the following into a microfuge tube:
20 µl reaction 50 µl reaction DNA 0.1 to 4 µg 0.1 to 4 µg 10x Digestion buffer 2 µl 5 µl Enzyme (as appropriate) Water (Rest of volume)
2. Add the enzyme (1-5u/µg DNA)
3. Incubate at recommended temperature for an hour.
4. Take 2 to 5 µl of the digested sample, add loading buffer, and run on agarose gel to check the result.
Tips:
1. DNA:
• For checking DNA, use 0.1 µg; (or 4-8 µl from a good DNA mini prep)
• For cloning, 4 µg DNA is enough
2. Buffer: besides the buffer that comes with the enzyme, buffers from other company can be used, too (as long as the contents are the same)
3. Enzyme: the maximum volume that an enzyme can be used is 1/10 of the total reaction volume (example: 2 µl for 10 µl reaction)
4. Incubation time: can be longer than 1 hr, but don’t do overnight digestion if you don’t have to (genomic DNA requires overnight digestion)
5. Gel: make sure to run the uncut DNA along with the digested DNA. And, always run a DNA marker!
Reference: Current protocols in molecular biology (3.1.1 - 3.1.2)
