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
- 10 ul 1:10 Taq DNA polymerase
- 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
