IGEM:Harvard/2007/Kevin's 91r Notebook

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(New page: *Step 1: Overnight culture from Perry's frozen stock of bacteria with LppOmpA plasmid. *Step 2: Plasmid prep of LppOmpA plasmid. *Step 3: Purification of plasmid. *Step 4: Design primers ...)
Current revision (16:53, 29 November 2007) (view source)
 
Line 4: Line 4:
*Step 4: Design primers for PCR.
*Step 4: Design primers for PCR.
*Step 5: Produce PCR product and run on gel.
*Step 5: Produce PCR product and run on gel.
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Result: Good bands for all three reactions, but signs of template present in all three.
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*Step 6: Topo cloning reaction: Mix PCR product and pTrcHis2-TOPO
*Step 6: Topo cloning reaction: Mix PCR product and pTrcHis2-TOPO
*Step 7: Transform mix into OneShot E.coli cells
*Step 7: Transform mix into OneShot E.coli cells
Line 9: Line 12:
*Step 9: Plasmid prep of culture
*Step 9: Plasmid prep of culture
*Step 10: Digestion with Nco1 and EcoR1 and run on gel.
*Step 10: Digestion with Nco1 and EcoR1 and run on gel.
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Result: Colonies grown because of template.  They either cut once (one site present in template) or cut twice (two close sites present on the template).
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Solution: Clonewell PCR product and select for the cut fragments.
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*Step 11: Clonewell PCR product
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*Step 12: Repeat steps 6-10.

Current revision

  • Step 1: Overnight culture from Perry's frozen stock of bacteria with LppOmpA plasmid.
  • Step 2: Plasmid prep of LppOmpA plasmid.
  • Step 3: Purification of plasmid.
  • Step 4: Design primers for PCR.
  • Step 5: Produce PCR product and run on gel.

Result: Good bands for all three reactions, but signs of template present in all three.

  • Step 6: Topo cloning reaction: Mix PCR product and pTrcHis2-TOPO
  • Step 7: Transform mix into OneShot E.coli cells
  • Step 8: Overnight culture of colonies to analyze positive clones
  • Step 9: Plasmid prep of culture
  • Step 10: Digestion with Nco1 and EcoR1 and run on gel.

Result: Colonies grown because of template. They either cut once (one site present in template) or cut twice (two close sites present on the template). Solution: Clonewell PCR product and select for the cut fragments.

  • Step 11: Clonewell PCR product
  • Step 12: Repeat steps 6-10.
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