IGEM:Harvard/2007/Kevin's 91r Notebook: Difference between revisions
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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 ...) |
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*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. | ||
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 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. | ||
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. | |||
Latest revision as of 13:53, 29 November 2007
- 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.
