From 2010.igem.org
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| - | {{Team:Alberta/beginMainContent}}
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| - | Protocol 17: PCR
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| - | Procedure:
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| - | * Before you start:
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| - | ** KEEP EVERYTHING ON ICE. Put Taq back into freezer as soon as you`re done with it. DON`T put back dNTP tubes.
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| - | ** Reserve a thermocycler and check what size of tubes it takes.
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| - | ** Making a master mix conserves expensive reagents, so try to always use one.
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| - | ** You may have to do dilutions of your reagents in order to make them usable for PCR (ex: primers, plasmid DNA...)
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| - | ** DON'T PUT PRIMERS OR TEMPLATE INTO THE MASTER MIX. ADD POLYMERASE TO THE MASTER MIX LAST (after your other tubes already have template DNA and primers in them)!
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| - | *To add into a tube:
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| - | {|
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| - | |PCR buffer || 5ul
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| - | |-
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| - | |10uM dNTPs || 1ul
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| - | |-
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| - | |50uM MgCl2 || 2ul
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| - | |-
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| - | |Forward primer || 2.5ul
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| - | |-
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| - | |Reverse primer || 2.5ul
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| - | |-
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| - | |1ng Template || 1ul
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| - | |-
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| - | |Taq polymerase || 0.5ul
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| - | |MilliQ water || 35.5ul
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| - | |TOTAL || 50ul
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| - | |}
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| - | * Program to run:
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| - | {|
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| - | |1. 94<sup>o</sup>C || 3 minutes
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| - | |-
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| - | |2. 94<sup>o</sup>C || 45 seconds
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| - | |3. 62<sup>o</sup>C || 30 seconds
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| - | |4. 72<sup>o</sup>C || 90 seconds
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| - | |5. Cycle steps 1 to 4 "30" times
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| - | |6. 72<sup>o</sup>C || 10 minutes
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| - | |}
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| - | [[Team:Alberta/Notebook/protocols| Back]]
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| - | {{Team:Alberta/endMainContent}}
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Latest revision as of 17:13, 26 October 2010
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