Team:UI-Indonesia/September

From 2013.igem.org





September

September 1st

  • Purification of PCR omega fragment
  • Making gene ruler
  • Running the purified product
  • Nanodrop the purified product : 110 ng/μL
  • Inoculating bacteria with pBluescript KS and pSB1C3 containg alpha fragment
  • Spreading BL21 and Top10 on agar plates with chloramphenicol antibiotic to check the optimum chloramphenicol concentration.
  • September 3rd

  • Nanodrop the large scale isolation product:
      pBluesript KS: 69.5 ng/μL
      Alpha fragment : 179.5 ng/μL
  • Restricting pBluescript KS using EcoRv
  • Nanodrop the restricted plasmid
  • Making competent cells
  • September 4th

  • Testing te chloramphenicol concentration
  • Testing the enzymatic activity from PstI
  • Making agar plates with different concentration of chloramphenicol
  • Running the result of enzymatic activity test
  • September 5th

  • Re-testing the enzymatic activity using different buffers
  • Running
  • Checking the chloramphenicol test result
  • Transforming:
      Omeg fragment-pBluescript KS
      (G4S)4 -pSB1C3 (ligated 3/9)
      Lox511-psB1C3 (ligated 3/9)
      Lox511-psB1C3 (ligated 25/8)
      Lox511-psB1C3 (ligated 25/8)
      (G4S)4 -pSB1C3 (ligated 25/8)
      pQE80L
      Re-ligating omega fragment-pSB1C3
  • September 6th

  • Restricting:
      Omega fragment + EcoRI
      pSB1C3
      Omega fragmetn +XbaI
      pBluscript KS + XbaI
      Alpha fragment + XbaI
  • Running
  • Nanodrop the purified digested plasmid
      Omega fragment + EcoRI = 7.2 ng/μL
      pSB1C3 = 3.8 ng/μL
      Omega fragment + XbaI = 7.7 ng/μL
      pBluescript KS + XbaI = 9.8 ng/μL
      Alpha fragment + Xba I = 0.35 ng/μL
  • Running
  • Inoculating bacteria with pSB1C3-alpha fragment in 100mL broth
  • September 9th

  • Large scale pSB1C3-alpha fragment isolation
  • Restricting pBluescript KS using EcoRV
  • Digesting linearized pSB1C3 and linearized omega fragment using PstI
  • Making 750mL LB Broth
  • Making agar plates
  • Digesting pSB1C3-alpha fragment
  • Purifying the digested using LMA
  • Blunting Lox511 and (G4S)4 linker
  • Running
  • September 11th

  • Small scale isolation
  • Running the small scale isolation product
  • September 13th

  • Re-run the small scale iolation product
  • Inoculate the bacteria containing pSB1C3=omega fragment
  • September 14th

  • Running small scale isolation product
  • PCR Colony
  • Large scale isolation
  • Re-ligating omega fragment with pSB1C3
  • September 15th

  • Running
      Digested pQE+PstI
      PQE
      Digested alpha fragment+PstI
      Alpha fragment
      Marker
      5
      8
      9
      15
      Large scale isolation pBluescript KS-omega