Standard Protocols
From 2006.igem.org
(Difference between revisions)
(→Presparation of Liquid Cultures) |
(→Presparation of Liquid Cultures) |
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# Add 15 ul of distilled water | # Add 15 ul of distilled water | ||
| - | === | + | === Preparation of Liquid Cultures === |
# Add 50 ml LB to 2 (or more) 250 ml conical flasks | # Add 50 ml LB to 2 (or more) 250 ml conical flasks | ||
# Add 50 microlitres of ampicillin to each flask | # Add 50 microlitres of ampicillin to each flask | ||
# Sterilise culture loop in a bunsen burner | # Sterilise culture loop in a bunsen burner | ||
| - | # Collect cells | + | # Collect cells from the sample plate using the sterile culture loop |
# Innoculate the flasks with the cells from the loop | # Innoculate the flasks with the cells from the loop | ||
# Sterilise the loop and repeat as necessary | # Sterilise the loop and repeat as necessary | ||
Revision as of 15:12, 12 July 2006
Contents |
Resuspension of Dry DNA
- Puncture a hole through the foil with a pipette tip
- Add 15 ul of distilled water
Preparation of Liquid Cultures
- Add 50 ml LB to 2 (or more) 250 ml conical flasks
- Add 50 microlitres of ampicillin to each flask
- Sterilise culture loop in a bunsen burner
- Collect cells from the sample plate using the sterile culture loop
- Innoculate the flasks with the cells from the loop
- Sterilise the loop and repeat as necessary
- Shake overnight at 37°C
Transformation into Competent Cells
- Take 1ul of suspended DNA and add to an aliquot of competent E-Coli cells (taken from the freezer at -80°C)
- Mix gently and leave in ice bucket for 10 to 30 minutes
- Place aliquot in heat block set to a temperature of 42°C for approximately 2 minutes (no longer than 3 minutes)
- Return to ice for a short period of time (approximately 3 minutes)
- Add 1 ml of LB liquid media
- Shake for about an hour at 37°C
- 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
- Incubate at 37°C overnight
- Remove from incubator and rest at room temperature for approximately an hour
- Move to refrigerator
Measurement of pH Change of lactose solution using E-Coli with and without lacZ
- Prepare a saturated overnight culture of E-Coli with and without lacZ
- 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
- Add 50 microlitres of ampicillin to each flask
- Incubate at 37°C with shaking for 30 to 60 minutes for cells to begin active growth.
- Measure and record the OD in each flask
- To each flask add 2.5 ml 20% w/v lactose and 50 microlitres of 90 mg/ml IPTG
- Aseptically measure the initial pH of each flask.
- 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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