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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 13:07, 28 January 2011
, 28 January 2011→Procedure
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::*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). | ||
::*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. | ||
#Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]]. | #Purify the PCR product using a [http://www.omegabiotek.com/products.php?CateID=14 kit] or [[Ethanol precipitation of nucleic acids|this protocol]]. | ||
