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Digestion
From 2008.igem.org
PRINCETON IGEM 2008
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Digestion Protocol
A Digestion is used to cut a plasmid at a specific site.
1. Use X units of enzymes per 1ug of DNA (over digesting by factor of X)
Usually we use over digest factor of 10 unless otherwise specified in the enzyme tech sheet. If over digestion results in star activity use 3X.
Total volume must be greater than or equal to 10X volume of enzyme.
3. Calculate the volume required for each
DNA µg = DNA volume * concentration
Enzyme volume = Enzyme unit/µl* # units = X [µl]
Buffer is dilution factor x dilution of the total volume.
[i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume]
6. Order of filling
• DNA
• Water
• Buffer
• Enzyme
7. Incubate for 3 hours at the specified temperature for the enzyme and deactivate at the appropriate temperature for 20 mins.
8. Please keep the buffer on ice at all times when out of -20C. Keep the enzyme in the benchtop coolers at all times when out of the -20C.
