Protocol 1: Produce LB Broth

Materials and Equipment

LB broth mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.

Time

Prep: 5 minutes
Run: 2 hours

Procedure

1. Weigh out 2g of LB broth mix in a weighing boat and add into a 250ml conical flask.
2. Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.
3. Swirl.
4. Stopper the flask with cotton wool and foil.
5. Label on autoclave tape. Format 'iGEM, Room Number'.
6. Move the flask(s) to the autoclave to be sterilised.

Protocol 2: Produce LB Agar

Materials and Equipment

LB Agar mix, sterile water, weighing scales, weighing boat, conical flask, measuring cylinder.

Time

Prep: 5 minutes
Run: 2 hours

Procedure

1. Weigh out 3.5g of LB agar mix in a weighing boat and add into a conical flask.
2. Measure 100ml sterile water into a measuring cylinder and dispense into the same conical flask.
3. Swirl.
4. Stopper the flask with cotton wool and foil.
5. Label on autoclave tape. Format 'iGEM, Room Number'.
6. Move the flask(s) to the autoclave to be sterilised.

Protocol 3: Make Overnight Starter Cultures

Materials and Equipment

Stripette, bunsen burner, sterile loop, falcon tubes, LB broth.

Time

Prep: 10 minutes
Run: 16 hours

Procedure

1. Use a stripette to take 5ml of LB broth from a 250ml conical flask.
2. Dispense into a falcon tube.
3. Sterilise a metal loop in a flame.
4. Take a culture from an agar plate using the sterile loop and put into one of the falcon tubes.
5. Agitate.
6. Replicate this using scrapings from a clean agar plate and a fresh tube to use as a positive control.
7. Put the tubes into the incubator overnight (37c, 150rpm) to grow up the cultures.

Protocol 4: Generate Chemically Competent E. Coli

Materials and Equipment

LB broth, starter culture, P1000, P200, pipette tips, incubator, cuvettes, spectrophotometer, Virkon, falcon tubes, ice, weighing scales, centrifuge, CaCl2, 20% glycerol, stripette, eppendorf tubes and -80°C freezer.

Time

Prep: 40 minutes
Run: 5 hours

Procedure

1. Grow Cells
   1. Take 1ml starter culture and add to 100ml LB broth.
   2. Incubate at 37°C, 150rpm.
   3. Check every hour by testing the optical density at 600nm (OD) using a spectrophotometer to determine whether enough cells are present in the culture.
   4. 0.600 OD is ideal, this is the point at which the cells are in the exponential growth phase.
   5. Take 1ml of culture into a cuvette to measure; dispose of this after use in Virkon.