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.


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