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From 2010.igem.org

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Contents 1 Notebook 1.1 July 29 (Thu) 1.2 July 31 (Thu) 1.3 August 2 (Mon) 1.4 August 3 (Tue) 1.5 August 4 (Wed) 1.6 August 5 (Thu) 1.7 August 6 (Fri) 1.8 August 7 (Sat) 1.9 August 8 (Sun) 1.10 August 9 (Mon)

Notebook

July 29 (Thu)

Attendance: Torigata, Takino, Teoh, Yasumoto, Kakuda, Saka, Tamura

1. Safety lecture for junior members.
2. Preparation of LB agar plates (26 Amp, 25 Kan, 25 Cam).

July 31 (Thu)

Attendance: Miyatake, Hirayama, Torigata, Teoh, Tadashi, Yasumoto, Kakuda, Saka

1. Meeting
   1. Summer project schedule
   2. List of genes to clone

August 2 (Mon)

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

1. Culture medium preparation
   1. LB agar plates (49 antibiotic-less plates)
   2. LB liquid medium (500 ml)
2. Competent cells preparation - Nojima Method
   1. SOB medium (MgCl2 not yet added) -> stored at 4˚C
   2. TB buffer -> stored at 4˚C
   3. 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)
   1. Preparation of glucose solution for making SOC medium.
   2. Inoculation of pre-culture SOB from o/n-incubated agar plate colonies.
   3. (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:

ID	Part Name	Resistance	Description
2-20J	<partinfo>BBa_K118023</partinfo>	A	C. fermi endocellulase Cen A coding
2-20H	<partinfo>BBa_K118022</partinfo>	A	C. fermi exocellulase Cex coding
1-2M	<partinfo>BBa_B0034</partinfo>	A	RBS
1-13D	<partinfo>BBa_B0010</partinfo>	A	terminator
1-1D	<partinfo>BBa_R0010</partinfo>	A	promoter
1-18F	<partinfo>BBa_E1010</partinfo>	K	RFP coding

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)

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

August 7 (Sat)

1. Miniprep
2. Transformation of construction plasmids

ID	Part Name	Resistance	Description
1-1C	<partinfo>pSB1A3</partinfo>	A	construction plasmid containing <partinfo>BBa_J04450</partinfo>
1-3A	<partinfo>pSB1C3</partinfo>	C	(" ")
1-5A	<partinfo>pSB1K3</partinfo>	K	(" ")

1. Meeting

August 8 (Sun)

1. Transfer to liquid culture medium

August 9 (Mon)

1. Miniprep of 1-1C
2. Restriction digests of 2-20H, 2-20J, 1-1D, 1-18F, 1-1C, 1-13D, 1-2M
3. Transfer of 1-3A, 1-5A colonies to solution culture (repeat)

   Yesterday's inoculated culture mediums contained the wrong antibiotics!