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Experiments & Protocols

Preparation of Competent E. coli cells

Grow cells to an OD600 of approximately 0.6.
Pour 50 mL of culture into a conical tube.
Centrifuge for 10 minutes at 3000 rpm at 4C.
Decant all media.
Resuspend pellet in 10 mL cold 0.1 M calcium chloride.
Incubate for 10 minutes on ice.
Centrifuge again for 10 minutes at 3000 rpm at 4C.
Decant the calcium chloride solution.
Resuspend the pellet in 5 mL cold 0.1 M calcium chloride/15% glycerol.
Aliquot around 200 ul of cells into microcentrifuge tubes.
Store at -80C immediately.

Transformation Protocol

Thaw tubes of cells on ice; have one tube designated as the control.
Mix DNA with cells (or diH2O if control) and incubate for 30 minutes on ice.
Heat shock at 42℃ for 30 seconds and on ice for 2 minutes.
Shake cells in SOC broth for 1 hour at 37℃, bring agar plates out of cold room to adjust to room temperature.
Deposit 200μL of each culture onto a plate, spread with glass spreader sterilized in ethanol, which is burned off. Use a flame to aid with sterility.
Leave plates for 10 minutes for culture to dry, then leave inverted in 37℃ incubator overnight.

Plasmid Isolation

Minipreps were performed using the MoBio UltraClean® Standard Mini Plasmid Prep Kit.

LB Broth

Mix 5 g tryptone, 2.5 g yeast extract, and 5 g NaCl in a microwaveable flask.
Fill with diH2O to 500 mL.
Autoclave in a tray that is covered in a thin layer of water.

SOC Broth

In one microwaveable flask, mix 10 g tryptone, 2.5 g yeast extract, 0.25 g NaCl, and 0.09 g KCl.
Fill with diH2O to 500 mL.
In a small container, mix 0.48 g MgCl2 and 1.23 g MgSO4 heptahydrate. Add a small amount of diH2O (<10mL).
In another small container, mix 1.80 g glucose with a small amount of diH2O (<10 mL).
Autoclave all containers in a tray that is covered with a thin layer of water.
Add the concentrated glucose and magnesium solutions to the 500 mL mixture.

LB Agar Plates

Mix 5 g tryptone, 2.5 g yeast extract, 5 g NaCl, and 7.5 g agar.
Fill with diH2O to 500 mL.
Autoclave in a tray that is covered in a layer of water. Store LB Agar until needed.
To prepare to make plates, microwave bottle of LB Agar until dissolved, then wait until bottle is cool enough to handle.
Add all necessary antibiotics in appropriate concentrations (100 mg/mL for ampicillin, 25 mg/mL for chloramphenicol, 50 mg/mL for kanamycin, 10 mg/mL for tetracycline).
Quickly pour mixture into as many plates as possible in a layer that covers the bottom. Use a nearby flame for sterility.
Cover plates and turn off flame when evaporation slows down.
Label and seal plates when the gel is solid, store in cold room.

Polymerase Chain Reaction

Mix 12.5 μL of Q5 Master Mix, 1.25 μL each of necessary forward and reverse primer, 2 μL of DNA sample, and 8 μL of diH2O in a PCR tube.
Run PCR program in thermal cycler, with annealing temperature and potential touchdown protocol set as needed.

Restriction Cloning

EcoRI and Pst used on pSB1C and the T7 gRNA vector
T4 Ligase used to ligate

Agarose Gel

Mix TAE buffer with 1% agarose (1 g per 100 mL) in microwaveable flask and microwave until dissolved.
Wait five minutes to cool. Add ethidium bromide solution needed for 0.2 μg/mL concentration.