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