Team:DTU-Denmark/Restriction protocol

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}}
 

Latest revision as of 19:19, 21 September 2011