Haynes Lab:Notebook/Engineering PC-TFs/2013/02/08

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Summary

  • * 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 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



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