From 2011.igem.org
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| - | {{:Team:DTU-Denmark/Templates/Standard_page_begin|Restriction/Digestion}}
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| - | == Restriction/Digestion ==
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| - | Materials:
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| - | * 25 µL DNA (but varies depending on your DNA conc.)
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| - | * 10 µL FD-buffer
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| - | * 1 µL restriction enzyme (1 & 2) (you can also use 2.5µL of each RE, which showed good results in previous digestions (AJ+PF))
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| - | * Using 1µL RE: 63ul ddH2O. Using 2.5µL RE: 60 µL ddH2O (Total volume of 100 µL)
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| - | # Turn the thermo block on 30 min before use. Mix everything – add the enzymes last. Leave for 2 hour at 37 C in thermo block/incubator.
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| - | # To deactivate the enzyme leave it at 65 C in a thermo block for 10 min.
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| - | # Keep on ice until its cooled down.
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| - | IMPORTANT: If you only use one RE on your plasmid
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| - | SAP treatment is required:
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| - | * Mix 50µL of the restricted DNA with 2.5µL SAP and 5µL SAP-buffer
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| - | * Put it in the incubator 37°C for 60 min
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| - | * 15 minutes on heat block 65°C to deactivate enzyme
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| - | * Place on ice
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| - | Enzyme selection for BioBricks digest
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| - | A BioBricks part:
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| - | ---E--X---Part---S--P---
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| - | Vector with upstream part - S,P
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| - | Downstream Insert - X,P
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| - | Upstream Insert - E,S
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| - | Vector with downstream part - E,X
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| - | E= EcoRI X= XbaI S= SpeI P= PstI
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| - | Enzymes should be kept in the icebox from the freezer at all times.
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| - | {{:Team:DTU-Denmark/Templates/Standard_page_end}}
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Latest revision as of 19:19, 21 September 2011
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