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From 2012.igem.org

Contents 1 General Protocol for Light App Experiments 1.1 Overnight Cultures 1.2 Culture Dilutions 1.3 Preparing a 96-Well Plate 1.4 96-Well Plate Culture 1.5 Using small (~2 in.) plates 1.6 Making Agar 1.7 Making buffered LB (pH = 6.0 or 8.0)

General Protocol for Light App Experiments

Overnight Cultures

1. Inoculate 3 mL culture tube of LB + 0.1M HEPES buffer pH = 8.0 + 6µL spectinomycin, 3µL kanamycin, and 3µL chloramphenicol with glycerol stock or a colony from a plate.
2. Cover it with aluminium foil and place it in 37°C shaker overnight.

Culture Dilutions

1. Get overnight culture from shaker and transfer 500 to 1000 µL of culture to a sterile 1.5mL tube.
2. Centrifuge at 13,000 rpm for 1 min.
3. Discard the supernatant in a waste bottle.
4. Resuspend in unbuffered LB - typically 500-1000µL.
5. Repeat the three steps above two more times (three washes, in total).
6. Dilute resuspended cells into unbuffered LB - 1:125 for soft agar. A dilution of 1:625 is sometimes better for other types of agar.

Preparing a 96-Well Plate

1. Melt agar in microwave and keep it in 37°C incubator for 20 minutes.
2. Calculate the amount of agar (see Making Agar section below) and add to sterile tube(s).
3. Add corresponding amount of antibiotics (1µL/mL chloramphenicol, 2µL/mL spectinomycin, 1µL/mL kanamycin).
4. Mix them without making bubbles.
5. Using a pipette, add the agar to each well (200µL/well). It takes ~20 minutes to cool down and solidify.
6. The plate can be saved by later by covering with film or foil and refrigerating at 4°C.

96-Well Plate Culture

1. Do the following steps in low light.
2. Pipet ~10uL diluted culture into wells in a 96-well plate.
3. Cover the plate with aluminium foil.
4. Wait for the culture to soak in. It takes about 10 minutes.
5. Program the Android light app and position the plate on tablet.
6. Place them in incubator for 15 hours.

Using small (~2 in.) plates

1. Melt agar in microwave and store it in 37°C incubator for ~20 minutes.
2. Pour necessary amount into sterile tube and add corresponding antibiotics. Each dish needs ~7mL agar.
3. Mix diluted bacteria at a ratio of 15:1000 into the agar. (the total dilution is therefore in the range of 1:40,000)
4. Mix without making bubbles.
5. Gently pour the bacteria+agar mixture into petri dishes, preferably in a darker place.
6. Cover plates with aluminium foil and wait for solidification (~25 minutes).
7. Program the Android light app and put the dishes, without lids, upside down on tablet.
8. Place them into 37°C incubator for 15 hours.

Making Agar

Example is for 100mL:

1. 100mL LB (pH=6.6, 0.1M HEPES), 50mg Ferric Amonium Citrate, 30mg S-gal, 1g agarose or low-melt agarose.
2. Mix and autoclave. Place in 4°C fridge.

Making buffered LB (pH = 6.0 or 8.0)

Example is for 100mL:

1. 100mL unbufferd LB, 2.383g (0.01mol) HEPES.
2. Adjust pH by using strong base and pH meter. (approximate amount is around 2-3 mL)
3. Mix and autoclave. Place in 4°C fridge.

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