Haynes Lab:Notebook/Engineering PC-TFs/2013/02/13

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  • Perform 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
gg2 pSB1A3 1.0
gg3 hPCD 1.0 BL01 1.0
10x ligase buffer 1.0
NEB T4 lgase 0.25
BsmBI 0.5
dH2O 5.25
Total 10.0

Could not perform bacterial transformation because SOC medium was in freezer. Vi performed BsmBI/T4 ligase and bacterial transformation for BL01 and hPCD and there were no colonies on plates.