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

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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. Reagent Vol. Thermal cycling [45°C, 2 min.; 16°C 5 min.] x25 60°C, 10 min. 80°C, 20 min. 4°C, ∞ 20 fmol (1 μL) of each DNA part up to 8.0 10x T4 ligase buffer (Promega) 1.0 T4 ligase (NEB) 0.25 BsmBI 0.5 dH2O 0.25   10.0 μL Reagent 1 2 3 gg2 pSB1A3 1.0 1.0 1.0 gg3 hPCD 1.0 1.0 1.0 BL02 1.0 --- --- BL03 --- 1.0 --- BL04 --- --- 1.0 10x ligase buffer 1.0 1.0 1.0 NEB T4 lgase 0.25 0.25 1.0 BsmBI 0.5 0.5 0.5 dH2O 5.25 5.25 5.25 Total 10.0 10.0 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.