Standard Protocols

From 2006.igem.org

Resuspension of Dry DNA

1. Puncture a hole through the foil with a pipette tip
2. Add 15 ul of distilled water

Preparation of Liquid Cultures

1. Add 50 ml LB to 2 (or more) 250 ml conical flasks
2. Add 50 microlitres of ampicillin to each flask
3. Sterilise culture loop in a bunsen burner
4. Collect cells from the sample plate using the sterile culture loop
5. Innoculate the flasks with the cells from the loop
6. Sterilise the loop and repeat as necessary
7. Shake overnight at 37°C

Transformation into Competent Cells

1. Take 1ul of suspended DNA and add to an aliquot of competent E-Coli cells (taken from the freezer at -80°C)
2. Mix gently and leave in ice bucket for 10 to 30 minutes
3. Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes)
4. Return to ice for a short period of time (approximately 3 minutes)
5. Add 1 ml of LB liquid media
6. Shake for about an hour at 37°C
7. Plate on agar containing the correct antibiotic, put approx. 100ul of cells on one plate, and the remainder on the other after centrifuging and resuspending the pellet
8. Incubate at 37°C overnight
9. Remove from incubator and rest at room temperature for approximately an hour
10. Move to refrigerator

Measurement of pH Change of lactose solution using E-Coli with and without lacZ

1. Prepare a saturated overnight culture of E-Coli with and without lacZ
2. Transfer 5 ml of culture to each of two 250 ml conical flasks (one with lacZ, one without) containing 50 ml of sterile LB medium
3. Add 50 microlitres of ampicillin to each flask
4. Incubate at 37°C with shaking for 30 to 60 minutes for cells to begin active growth.
5. Measure and record the OD in each flask
6. To each flask add 2.5 ml 20% w/v lactose and 50 microlitres of 90 mg/ml IPTG
7. Aseptically measure the initial pH of each flask.
8. Return the flasks to the 37 C shaker. Retest the pH after 15, 30, 45, 60 minutes, and then at further intervals depending on the results of these assays.

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