Smolke:Protocols/Insert prep

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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...)
Line 7: Line 7:
**10 ul Rev primer
**10 ul Rev primer
**250 ng plasmid
**250 ng plasmid
 +
**10 ul 1:10 Taq DNA polymerase
*Taq amplifies at 1000-2000 bp/min, so adjust extension time accordingly
*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
*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
*Purify and concentrate PCR products by phenol-chloroform extraction and ethanol precipitation
*Resuspend inserts to appropriate volume for digestion reaction
*Resuspend inserts to appropriate volume for digestion reaction

Revision as of 13:33, 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
    • 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
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