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Week2:August 1 - August 7
August 2 (Mon)
Attendance: Torigata, Takino, Teoh, Tadashi, Yasumoto, Saka
- Culture medium preparation
- LB agar plates (49 antibiotic-less plates)
- LB liquid medium (500 ml)
- 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
- 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
- OD measurements throughout the day till required OD (0.3~0.7) was obtained.
- Completion of competent cells according to protocol.
August 5 (Thu)
Attendance: Nakamura, Yasumoto, Saka, Kakuda, Takino
- Transformation of Registry parts:
| ID | Part Name | Resistance | Description
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| 2-20J | <bbpart>BBa_K118023</bbpart> | A | C. fermi endocellulase Cen A coding
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| 2-20H | <bbpart>BBa_K118022</bbpart> | A | C. fermi exocellulase Cex coding
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| 1-2M | <bbpart>BBa_B0034</bbpart> | A | RBS
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| 1-13D | <bbpart>BBa_B0010</bbpart> | A | terminator
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| 1-1D | <bbpart>BBa_R0010</bbpart> | A | promoter
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| 1-18F | <bbpart>BBa_E1010</bbpart> | K | RFP coding
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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.
August 6 (Fri)
Attendance: Nakamura, Saka, Yasumoto, Takino, Teoh
- 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
- Colonies transferred to 3ml LB growth medium & incubated o/n at 37˚C
August 7 (Sat)
Attendance: Nakamura, Saka, Yasumoto, Takino
- Miniprep of o/n-incubated colonies (2-20J, 2-20H, 1-2M, 1-13D, 1-1D, 1-18F) using Sigma-Aldrich Plasmid Miniprep Kit
- Transformation of construction plasmids
| ID | Part Name | Resistance | Description
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| 1-1C | <bbpart>pSB1A3</bbpart> | A | construction plasmid containing mRFP coding device (<bbpart>BBa_J04450</bbpart>)
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| 1-3A | <bbpart>pSB1C3</bbpart> | C | (" ")
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| 1-5A | <bbpart>pSB1K3</bbpart> | K | (" ")
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- Meeting
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July
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Su |
Mo |
Tu |
We |
Th |
Fr |
Sa |
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1 |
2 |
3 |
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5 |
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11 |
12 |
13 |
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18 |
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21 |
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| Week1 |
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29 |
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31 |
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