Difference between revisions of "Team:Tec-Monterrey/Notebook"

Pre-Interlab: Transformation Practice (p. 25, 07/13/18) Starting time: 11:45 am Antibiotic stock solutions (Carlos) Ampicillin 100 mg/ml Chloramphenicol 35 mg/ml Kanamycin 35 mg/ml LB Medium (without agar) (Jesús) 25 g/L, 500 ml of medium LB Medium (with agar) (Jesús) 25 g/L, 500 ml of medium 15 g/L of agar, 500 ml of medium Medium LB and silica spheres are put inside autoclave 121°C for 15 minutes Materials left inside fume hood, with 15 min. of UV light Bain-marie prepared, 42°C Pre-Interlab: Transformation Practice (p. 25, 07/13/18) Transformations (Samantha) 4 Petris, 23K + antibiotic, 8P + antibiotic, 23K w/o antibiotic, competent w/o antibiotic Positive control: 8P, dish 3, kit 2018: BBa_I20270 (GFP) 1: 23K, dish 3, kit 2018: BBa_I763007 (RFP) DNA from plates to Eppendorfs 1: 50 μL CC, 1 μL 23K 2: 50 μL CC, 1 μL 23K 3: 50 μL CC, 1 μL 8P 4: 50 μL CC Plates (Victor) 20 ml LB with agar for all plates 20 μL of Chloramphenicol when needed Heat Shock (Samantha) 1-4: 1 minute, 42°C, then 5 minutes on ice + 200 μL LB to each tube Note: iGEM protocol says 450 ml of SOC, but instead we used 200 μL of LB Incubations 37°C and 220 rpm, during 1 hour (6:03-7:03 pm) Interlab: Phase 1 and Preparations (p. 26-27, 07/15/18-07/16/18) SOC medium preparation (NCb BioLabs, Thermo Fisher) 2% Tryptone 0.5% yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 20 mM Glucose SOB medium preparation 2% Tryptone 0.5% yeast extract 10 mM NaCl 2.5 mM KCl 10 mM MgCl2 10 mM MgSO4 Buffer CCMB80 1L preparation 10 mM KOAc ph 7.0 80 mM CaCl2 20 mM MnCl2 10 mM MgCl2 Growth In 200 ml of SOC Notes: iGEM protocol says 250 ml SOB, instead 200 ml were used There’s less overnight, so initial absorbance will be less than 0.1 Optical density 3:00 pm, 0.3 3:52 pm, 0.465 Note: For buffer resuspension, 20 ml were used instead of 80 ml RFP transformation after 16 hours 12 hours: 1-2 colonies, low expression 16 hours: 3-4 colonies, medium expression Second competents batch Previous results were successful, so iGEM (buffer CCMB80) protocol will be repeated. Absorbance before first resuspension: 0.482 Resuspension in 20 ml of buffer Absorbance after resuspension: 0.444

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