From 2007.igem.org

Contents 1 TATA_INSA, TATB_INSB, TATD_IDE and OmpRBS GEL check after PCR 2 re-culture pOmpC and pCinRHSL 3 digestion for assembly 3.1 System 1 3.2 System 2

TATA_INSA, TATB_INSB, TATD_IDE and OmpRBS GEL check after PCR

• from left to right
  • lane 1: 100bp ladder
  • lane 2-4: TATA_INSA (PCR 1+2)
  • lane 5-7: TATB_INSB (PCR 3+4)
  • lane 8-12: OmpRBS (PCR 6)
  • lane 13: 1kb ladder
  • lane 14-16: TATD_IDE (PCR 5+7)
  • lane 17: 100bp ladder
  • lane 18: OmpRBS (PCR 6)

re-culture pOmpC and pCinRHSL

• Because of the low concentration of pOmpC and pCinRHSL, the liquid culture is performed again to gain larger concentration
• concentrations
  • pCinRHSL (0.05 ug/uL by pett, 0.05 and 0.08 by 海威)
  • pOmpC #1 (run out off in previous experiment)
  • pOmpC #2 (0.02 ug/uL by pett, 0.04 and 0.05 by 海威)
  • pOmpC #3 (0.04 and 0.23 by 海威)
  • pOmpC #4 (0.04 and 0.08 by 海威)

digestion for assembly

System 1

• pOmpC + TATA_INSA
  • pOmpC (re-cultured, 10/2; S.P. digestion, 10/3)
  • TATA_INSA (X.P. digestion, 10/2)
• TATB_INSB + CinR+HSL+D-term
  • TATB_INSB (E.S. digestion, 10/2 (it is not correct!!! TATB_INSB does not have EcoRI site)
  • CinR+HSL+D-term (E. digestion, 10/2)
  • CinR+HSL+D-term (X. digestion, 10/3)
• (pOmpC + TATA_INSA) + (TATB_INSB + CinR+HSL+D-term)
  • (pOmpC + TATA_INSA)
  • (TATB_INSB + CinR+HSL+D-term)

System 2

• pCinRHSL + OmpRBS
  • pCinRHSL (re-cultured, 10/2; S.P. digestion, 10/3)
  • OmpRBS (X.P. digestion, 10/2)
• (pCinRHSL + OmpRBS) + TATD_IDE
  • (pCinRHSL + OmpRBS)
  • TATD_IDE
• [(pCinRHSL + OmpRBS) + TATD_IDE] + D-term
  • [(pCinRHSL + OmpRBS) + TATD_IDE]
  • D-term