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From 2010.igem.org

Contents 1 August 16 (Mon) 2 August 17 (Tue) 3 August 18 (Wed) 4 August 19 (Thu) 5 August 20 (Fri) 6 August 21 (Sat)

August 16 (Mon)

1. Restriction digests
   • 1-13D,　1-2M,　1-1D,　1-18F with EcoRI, PstI to check for point mutation(s) affecting XbaI site
   • 2-20J with XbaI, PstI to check/confirm XbaI activity
2. Gel electrophoresis of digests
   • (RESULTS?)
3. Colony pick-up & transfer to solution culture (repeat; 2 each): 1-13D,　1-2M,　1-1D,　1-18F
   • 3ml LB liquid medium; 3μl Amp or 0.6μl Kan solution added

August 17 (Tue)

1. Mini-prep of 1-13D,　1-2M,　1-1D,　1-18F parts inoculated yesterday (2 of each)
2. 'Cut check' (restriction digest + gel electrophoresis to confirm insert) of the miniprepped parts:
   • 1-1D, 1-18F with EcoRI, SpeI
   • 1-13D, 1-2M with XbaI, PstI
   • (RESULTS?)
3. Transformation of secretion tag parts using 25μl of competent cells each(See Table 3)

Table 3

ID	Part Name	Resistance	Description
2-22P	<bbpart>BBa_K103006</bbpart>	A	OmpA outer membrane protein + linker
1-2J	<bbpart>BBa_J32015</bbpart>	A,K	PelB leader sequence

August 18 (Wed)

iGEM Japan Meet-Up in Kyoto Attendance: Nakamura, Yasumoto, Saka, Kakuda

August 19 (Thu)

1. Transfer of 2-22P, 1-2J to solution culture
2. Gel electrophoresis of digests from 'cut check' products from Tuesday
   • repeat run, but each digest together with undigested plasmid DNA)
   • 2% agarose gel instead of the usual 1%
   • (RESULTS?)
3. Gel electrophoresis of 1-1D digest only
   • (RESULT?)
4. Multiple restriction digests of 1-1D to check for problems at restriction sites
   • tried the following: EcoRI only; SpeI only; EcoRI + SpeI
5. Night: miniprep of 2-22P, 1-2J inoculated in the morning

August 20 (Fri)

1. Gel electrophoresis of 1-1D and its digests
   • (RESULTS?)
2. 'Cut check' of parts miniprepped the night before
   • both 2-22P & 1-2J cut with XbaI, PstI
   • enzyme inactivation at 80°C, 20min
   • (RESULTS?)
3. Restriction digest of 2-20J (WHICH ENZYMES?)
4. Ligation according to 3A assembly method: 2-20J or 2-20H + 2-22P or 1-2J; 1-3A as vector
   • reaction mixture: 2μl of each plasmid, 2μl ligase buffer, 1μl T4 DNA ligase, water to make 20μl total
   • reaction at room temperature for 10min; ligase inactivation at 80°C for 20min
5. Transformation of ligated parts using 50μl of competent cells each; 2μl ligation product; 150μl Chloramphenicol spread on agar plates before inoculation with pre-incubation mix

August 21 (Sat)

1. Transfer of white (non-RFP) colonies from yesterday's transformation to 3ml LB liquid medium added with 1μl Cam solution
   • we later realized that the upstream and downstream parts had been mixed up in these ligations, so the ligation products (and these corresponding solution cultures) were discarded
2. 3A assembly ligation: 1-1D as upstream, 1-2M as downstream, 1-3A as vector
   • ligation product designated as 001; Chloramphenicol resistance
   • same ligation mix composition as yesterday's
3. Transformation of 001 with pre-incubation for 1.5hr instead of 1hr