User:Karmella Haynes/Notebook/BioBrick cloning/2013/01/09

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01/09/13

  • Golden Gate assembly: hPCD + BL01 + pSB1A3
  • Violacein: plates from Tabor; incubate o/n



Golden Gate Assembly

  • Use purified, measured PCR products from 1/05/13
  • Use gg2 (pSB1A3), gg3, gg4; not gg1 (V0120) since it has BsmBI sites
  • Dilutions
DNA length (bp) ng/μL Vol. + dH2O
gg2 pSB1A3 2000 67.7 7.7 12.3
gg3 hPCD 186 22.5 2.1 17.8
gg4 BL01 2520 55.9 11.7 8.3


  • Golden Gate Reactions
  1. hPCD + BL01 + pSB1A3
  2. pSB1A3
Reagent 1 2
gg2 pSB1A3 1.0 1.0
gg3 hPCD 1.0 ---
gg4 BL01 1.0 ---
10x ligase buffer 1.0 1.0
NEB T4 lgase 0.25 0.25
BsmBI 0.5 0.5
dH2O 5.25 7.25
  10.0 10.0

Thermal cycler

  • [45°C, 2 min.; 16°C, 5 min.] x25
  • 60°C, 20 min.
  • 80°C, 20 min.



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