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

Pre-Interlab: Transformation Practice (p. 25, 07/13/18)

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, 07/16/18)

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 the first resuspension: 0.482
• Resuspension in 20 ml of buffer
• Absorbance after resuspension: 0.444

Interlab: Plate 7 Transformations (p. 28-29, 07/17/18-07/18/18)

DNA Resuspension (Samantha)

• With respect to the wells on plate 7

Competent cells

• 50 μL to each Eppendorf duplicate

Resuspended DNA

• 2 μL were added to each Eppendorf duplicate

Colonies