Jessica Karen Wong/Notebook/2007-7-6

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To Do

  • Redo E0240 PCR w/ vent and phusion
  • Ligate, transform T9002 and 3k3
  • Remake overnight cultures of I2056


  • Redid 2 100ul prep pcr's with Vent and Phusion
  • Just realized that we completely skipped the ligation and transformation with backbone
  • Discarded PCR product
  • Did two way ligations E0240-3K3 and E0240-1AK3 (used 4.5ul of each E0240 and backbone)
  • Transformed 3 ul of each ligation and plated


  • Ligated T9002-D and T9002-S with 3K3 (Double and Sequentially digested)
  • Transformed using 3ul of ligation
  • Plated on Kan plates


  • Got sequencing back and all samples do not contain R0040
  • Religated R0040-I13401-1AK3, R-I-1AT3, R-I-1AC3 (3ul each part)
  • Transformed 3 ul of each ligation and plated
  • Primer to PCR in R0040 came in
  • Doing a 100ul prep PCR of the #4 colony of I2055 on 1AK3
  • Using Vent at 53C (1:30 extension time)

From now on should use Vent for all Preparatory PCR reactions


  • Overnight liquid cultures did not grow
  • Made new overnights from actual colony instead of cell suspension