Smolke:Protocols/Insert prep: Difference between revisions
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(New page: *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 amplifie...) |
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Revision as of 10:33, 15 October 2009
- 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
