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Team:Wageningen UR/Notebook/Proj1/July
From 2011.igem.org
July 5
We created and isolated the following parts: C0061-B0015 & F2621-E0422 & F2621-I0460 & R0062-I0460.
July 6
Digestions and ligations :)
July 8
Colony PCR from the ligations made on July the 6th.
July 11
More digestions and subsequent ligations.
We also created and isolated the following parts: K182102-C0061-B0015 & R0062-I0460.
July 13
Colony PCR from the ligations made on July the 11th. BioBrick parts were digested using different combinations of restriction enzymes and run on a gel.
July 14
Ligations were performed using the digestions from the 13th.
July 15
Colony pcr to verify the ligations of the 14th and subsequently liquid cultures of the positive colonies were made. We also created and isolated the following parts: K318511-S01003 & K182102-C0061-B0015-R0062-I0460 & R0040-K082014 & F2621-K082014-F2621 & F2621-E0422-F2621-K082014 & R0040-E0422 & F2621-K082014-F2621-E0422 & E0422-F2621-K082014 & & F2621-E0422-F2621-I0460 & R0040-K082014
July 20
Digestions, ligations, transformations.
July 22
PCR screen of the ligations from July 20th.
July 23
Digestions and ligations.
July 25
Transformations of the ligations from the 23rd.
July 26
We created and isolated the following parts: K182102-c0061-B0015 & R0062-I0460 & F2621-K082014 & F2621-I0460. More digestions, ligations and transformations.
July 27
Colony pcr to verify yesterday’s ligations.
July 28
Digestions, ligations, transformations.
July 29
Colony PCR and making liquid cultures
Also making new digestions and ligations.
July 30
Isolating the parts from the liquid cultures made on the 29th. Besides that, we also inoculated 500 mL erlenmeyers with 100 ml LB + 100 ug/ul. E. coli was grown overnight on LB + amp plates and directly used as inoculum. All cultures were done in duplo. E. coli strains used were: ptet-GFP R0062-GFP F2621-GFP RBS-GFP
Cultures were put in a shake incubator at 30°C, 300 rpm. Temperature was set to 30 degrees as GFP is degraded at higher temperatures. The OD600s of every culture were measured 2 hours after inoculation and subsequently each hour, by taking 1 mL samples from the culture. the OD was measured with a Hitachi U1500 spectrophotometer. LB+amp was taken as blank. Fluorescence was measured in a 96-wells plate on a fluorescent plate reader (Molecular Devices, M2 (toxicology). First blank measurements were done with 96 wells plates containing 50 ul LB+amp. The output corresponding with the specific wells was saved and used for further calculations.
OD600 data presented below
| Sample | no. 1 (2 hours) | no. 2 (2 hours) | no. 1 (4 hours) | no. 2 (4 hours) | no. 1 (6 hours) | no. 2 (6 hours) |
|---|---|---|---|---|---|---|
| RBSGFP | min 0.001 | min 0.001 | 0.021 | 0.004 | 0 | 0 |
| R0062 | min 0.016 | min 0.011 | 0.001 | 0.020 | 0 | 0 |
| ptetGFP | min 0.006 | min 0.004 | 0.011 | min 0.003 | 0 | 0 |
| F2621GFP | min 0.014 | min 0.003 | 0.009 | 0.011 | 0 | 0 |
