NYMU Taipei/Lab Notes/2007 9 11

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(Difference between revisions)
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*** ligase 1uL
*** ligase 1uL
*** add water to make total volume as 10 uL
*** 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, 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)

Revision as of 15:25, 11 September 2007

  • 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 YEA ligation protocol, it requires
      • ligation buffer A 1uL
      • ligation buffer B 1uL
      • 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, 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)
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