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.