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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 15:08, 25 January 2011
, 25 January 2011→Procedure
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#[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [[Miniprep/Qiagen kit|a kit]] or [[Miniprep/GET buffer|this protocol]] (30 mins). | #[[miniprep|Miniprep]] both "insert" and "vector" from their respective cultures using [[Miniprep/Qiagen kit|a 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). | ||
::*Use a high-fidelity polymerase (e.g. pfu Turbo or | ::*Use a high-fidelity polymerase (e.g. pfu Turbo or [http://www.neb.com/nebecomm/products/productM0254.asp 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). | ||
#[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | #[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | ||
