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Richard Lab:Amplified insert assembly: Difference between revisions
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Richard Lab:Amplified insert assembly (view source)
Revision as of 12:21, 28 January 2011
, 28 January 2011→Procedure
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#[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [http://www.omegabiotek.com/products.php?CateID=11 kit] or [[Miniprep/GET buffer|this protocol]] (30 mins). | #[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [http://www.omegabiotek.com/products.php?CateID=11 kit] or [[Miniprep/GET buffer|this protocol]] (30 mins). | ||
#PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away). | #PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away while the insert PCR is cycling). | ||
::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent). | ::*Use a high-fidelity polymerase (e.g. pfu Turbo or Vent). | ||
::*Use the same primers you use for colony PCR (Annealing Temp of 55°C). | ::*Use the same primers you use for colony PCR (Annealing Temp of 55°C). | ||
