From 2010.igem.org
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| - | Protocol 5: Restriction Digest
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| - | What you will need:
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| - | * Restriction enzyme buffer
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| - | ** Buffer information can be found at www.NEB.com using the NEBtools
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| - | * Restriction enzymes
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| - | * milliQ water
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| - | * DNA to be digested
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| - | Procedure:
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| - | * Select a restriction enzyme buffer that is appropriate for BOTH of the enzymes you are using. See information sheets at front of lab for correct buffer and concentration.
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| - | * The total volume of all enzymes in the reaction should be less than 10% of the final reaction volume. Enzymes usually are supplied at 10U/ul and 1ul will be more than enough for our digests.
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| - | * Add components in the following order:
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| - | **Water
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| - | **Buffer
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| - | **Bovine Serum Albumin-BSA (if needed)
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| - | **DNA
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| - | **Enzyme I
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| - | **Enzyme II
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| - | An example of a typical 25 ul reaction would be
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| - | {|
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| - | |milliQ water || 15.5 ul
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| - | |-
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| - | |10x NEBuffer || 2.5 ul
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| - | |-
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| - | |DNA (200 ng/ul) || 5.0 ul
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| - | |-
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| - | |XbaI || 1.0 ul
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| - | |-
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| - | |PstI || 1.0 ul
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| - | |}
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| - | Incubate at 37 C for one hours (longer is okay too).
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| - | ==Notes==
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| - | FastDigest (by Fermentas) enzymes use a single uniform buffer, and claim to work in 5 min.
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Latest revision as of 17:16, 26 October 2010
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