Team:Osaka/week2

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Contents

August 2 (Mon)

Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka

  1. Culture medium preparation
    • LB agar plates (49 antibiotic-less plates)
    • LB liquid medium (500 ml)
  2. Competent cells preparation - Nojima Method
    • SOB medium (MgCl2 not yet added) -> stored at 4˚C
    • TB buffer -> stored at 4˚C
    • Single-colony streaking of E. coli (strain DH5α) on 2 LB agar plates -> 37˚C incubation o/n

Tomorrow we shall prepare the remainder of the reagents and inoculate SOB with o/n-incubated E. coli.

August 3 (Tue)

Attendance: Nakamura, Kakuda, Saka, Yasumoto, Teoh

  1. Competent cells preparation (continued)
    • Preparation of glucose solution for making SOC medium.
    • Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
    • (Night) Transfer from pre-culture to growth culture.

August 4 (Wed)

Attendance: Nakamura, Saka, Kakuda, Yasumoto, Torigata, Teoh

  1. OD measurements throughout the day till required OD (0.3~0.7) was obtained.
  2. Completion of competent cells according to protocol.

August 5 (Thu)

Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino

  1. Transformation of Registry parts (See Table 1).

Note: 'ID' is an internal identifier used by wet work members to simplify labeling etc. In the case of iGEM distribution parts it usually refers to the plate location.

Table 1
IDPart NameResistanceDescription
2-20J<bbpart>BBa_K118023</bbpart>AC. fermi endocellulase Cen A coding
2-20H<bbpart>BBa_K118022</bbpart>AC. fermi exocellulase Cex coding
1-2M<bbpart>BBa_B0034</bbpart>ARBS
1-13D<bbpart>BBa_B0010</bbpart>Aterminator
1-1D<bbpart>BBa_R0010</bbpart>Apromoter
1-18F<bbpart>BBa_E1010</bbpart>KRFP coding

August 6 (Fri)

Attendance: Nakamura, Saka, Yasumoto, Takino, Teoh

  1. Colony check
    • All transformed cells produced colonies!
    • Non-transformed cells (negative controls) did not grow on Amp, Kan or Cam plates -> confirmed lack of natural antibiotic resistance
  2. Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C

August 7 (Sat)

Attendance: Nakamura, Saka, Yasumoto, Takino

  1. Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
  2. Transformation of construction plasmids (See Table 2)
  3. Meeting
Table 2
IDPart NameResistanceDescription
1-1C<bbpart>pSB1A3</bbpart>Aconstruction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>)
1-3A<bbpart>pSB1C3</bbpart>C(" ")
1-5A<bbpart>pSB1K3</bbpart>K(" ")