Haynes Lab:Notebook/Engineering PC-TFs/2013/02/14

Summary

• * Performed BsmBI/ T4 ligase mediated assembly
• BsmBI cuts the DNA fragments and creates complementary overhangs.
• Complementary sticky ends anneal via base pairing.
• T4 ligase seals gaps in the phosphodiester DNA backbone.

Bacterial transformation

• Added total volume (10.0 μL) to 50 μL chemically competent cells (e.g., BL21) in a 2.0 mL tube.
• Incubated on ice for 2 min., heat shock at 42°C for exactly 45 sec., and placed on ice.
• Added 800 μL sterile SOC medium.
• Grow with shaking at 37°C for 30 min.(Taped tubes to the shaker rack.)
• Pelleted the cells at top speed in a microcentrifuge for 3 min. at room temperature.
• Discarded the supernatant. Resuspended the cells in 100 μL LB + antibiotic.
• Plate cells on pre-warmed LB agar + antibiotic. (I plated 100 μL of the cells. Then put beads on plates and shook.) Protocol didn't specify.) Grow overnight at 37°C.
• Check results tomorrow.

There were no colonies on the plates. It's possible that the concentration of the ligation product is to low, so I will try to amplify.