From 2007.igem.org

• Fresh back
  • two different PCR (INS_A and INS_B) have been done by 海威 (9/5, 2007) and 惇茹 (9/8, 2007)
  • we had failed three times for construction of pOmpC + INS_A using 惇茹's INS_A and following three different ligation protocol
    • insert is INS_A and vector is pOmpC
    • insert (1 uL) vs. vector (1 uL) at 09/09, 2007: the quantity are 6ug = 6,000 ng vs. 13ug = 13,000 ng (original measure)
    • insert (2 uL) vs. vector (1 uL) at 09/10, 2007: the quantity are 12ug = 12,000 ng vs. 13ug = 13,000 ng (original measure)
    • insert (3 uL) vs. vector (1 uL) at 09/10, 2007: the quantity are 18ug = 18,000 ng vs. 13ug = 13,000 ng (original measure)
    • a trivial failure (absent of vector part) at 09/08, 2007
  • According to [http://www.yeastern.com/index.html YEA (益生)] ligation protocol, it requires
    • ligation buffer A 1uL
    • ligation buffer B 1uL (Note: only needed in blunt end ligation)
    • vector 50 ng or 100 ng (proposed by pett)
    • insert 300 ng
    • ligase 1uL
    • add water to make total volume as 10 uL
  • Because the mistake on calculation of part concentrations, the concentration is over-estimated (concentration is far lower than we expected)
    • Obviously, the concentration of INS_A from 惇茹 (9/8, 2007) is too low
    • Thus, we use GEL to check the 海威's (9/5, 2007) PCR product (10uL), the band seems good
• Remark
  • the key of failure might be the insert concentration is too low
  • the corrected concentration of pOmpC (vector) should be 0.06 ug/uL (60ng/uL) and INS_A (insert) should be 0.13 ug/uL (130ng/uL)
  • to fulfill the requirement of ligation protocol, the volume of vector should be 1.66 uL and volume of insert should be 2.3 uL
  • However, we do not believe the concentration is correct and it might be very low (because the OD is almost zero)
  • To correctly measure the concentration and increase the production of INS_A through PCR, GEL extraction and digestion are two ways to successful construct
  • PCR might be tested and varied by different annealing temperature
  • Beside, our vector (pOmpC) has been successfully ligated with other Biobrick. It should be O.K. (However, we use ligation kit from different company)