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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 21:22, 18 March 2011
, 18 March 2011→Procedure
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#PCR the "insert" plasmid (This will take about 2 hrs, but start the vector digest right away while the insert PCR is cycling). | #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 | ::*Use the same primers you use for colony PCR (Annealing Temp of 55-60°C). | ||
::*Only run 25-30 cycles as this will help ensure high fidelity. | ::*Only run 25-30 cycles as this will help ensure high fidelity. | ||
#[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | #[[Richard Lab:Restriction Digest|Digest]] the "vector" for 2 hours. | ||
