Digestion with Restriction Enzymes
From 2007.igem.org
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== Vector digestion == | == Vector digestion == | ||
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 | 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 | ||
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Calculate the volume required for each | Calculate the volume required for each | ||
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DNA µg = DNA volume * concentration | DNA µg = DNA volume * concentration | ||
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Enzyme volume = Enzyme unit/µl* # units = X [µl] | 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] | Buffer is dilution factor x dilution of the total volume. [i.e. for 10X over digest - buffer is 10%, 3x - 30% of total volume] | ||
Order of filling | Order of filling | ||
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DNA | DNA | ||
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Water | Water | ||
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Buffer | Buffer | ||
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Enzyme | Enzyme | ||
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Incubate for 3 hours at the specified temperature for the enzyme and deactivate at the appropriate temperature for 20 mins. | Incubate for 3 hours at the specified temperature for the enzyme and deactivate at the appropriate temperature for 20 mins. | ||
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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. | 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. | ||
Latest revision as of 02:59, 27 October 2007
Vector digestion
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
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] Order of filling
DNA
Water
Buffer
Enzyme
Incubate for 3 hours at the specified temperature for the enzyme and deactivate at the appropriate temperature for 20 mins.
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.
