Smolke:Protocols/Insert prep

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  • Prepare 5 x 100-ul PCR reactions for each insert
    • 400 ul water
    • 50 ul 10x Taq buffer
    • 16 ul 5 mM MgCl2
    • 10 ul dNTP
    • 10 ul Fwd primer
    • 10 ul Rev primer
    • 250 ng plasmid
  • Taq amplifies at 1000-2000 bp/min, so adjust extension time accordingly
  • After PCR, combine products from the five tubes, and run 5 ul on a gel to verify product length
  • Purify and concentrate PCR products by phenol-chloroform extraction and ethanol precipitation
  • Resuspend inserts to appropriate volume for digestion reaction
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