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Wet Lab: Part Measurements and Characterisation
Characterisation of constitutive promoters:
- You have been provided with 2 x 250ml conical flasks containing 100ml of sterile LB media. Aseptically, add 5 ml of over-night culture (Positive control - Cells containing the constitutive promoter construct) with a 5ml sterile pipette to one of the flasks. Then add 100 µl of 50mg/ml Kanamycin (1000 X stock). Swirl to mix. Label this flask with your initials and a “+” sign.
- Repeat step 1 by aseptically adding 5 ml of over-night culture (Negative control – Cells without plasmid) to the other flask. Then add 200µl of 5mg/ml Tetracycline (500 X stock). Swirl to mix. Label this flask with your initials and a “–” sign.
- Take out 1ml from each flask with a p1000 Gilson and transfer to 1ml cuvettes. The sample from the positive control flask should be labelled “+” and the sample from the negative control flask should be labelled “–”. One of the pair should measure the OD600 of both samples and record the readings in Table 1 on page 3. Also, make a note of the time as this will be taken as time zero when you take further OD readings at 1 hour intervals. The other of the pair should also take out 1ml from each flask but transfer the aliquots to Eppendorf tubes labelled “0h+” and “0h-” where the 0 represents the time in hours. Also, label the tubes with your initials. Leave these tubes in your ice bucket as these will be used for fluorescent measurements later on in the day.
- Take out another 1 ml from each flask, transfer to Eppendorf tubes (label “+” & “–” and don’t forget to include your initials on the tubes), then spin the cells down in a microfuge at 1,000rpm for 5 mins. Make sure your tubes are balanced!
- Once you have loaded your samples in the microfuge, put your 2 x 250ml conical flasks in the 37°C shaking incubator.
- At the end of the 5 minute spin in the microfuge, remove the supernatant from each tube and store the pellets at -20°C overnight.
- At 1hour intervals, repeat step 3, 4, 5 and 6 (taking & replacing your 2 x 250ml conical flasks from the 37°C shaking incubator) until you have 6 OD600 readings (12 in total) and 6 positive control and negative control samples in your ice bucket for fluorescent measurements + 12 samples (6 neg. control and 6 pos. control) in the -20°C freezer for the next day. (You have been provided with 6 cuvettes so you’ll need to rinse these out with distilled water and re-use them. 10ml of LB medium has also been provided for you as you’ll probably have to dilute your samples when the OD readings become too high ~ 0.8. For the fluorescence samples, label your Eppendorf tubes “1h+” and “1h-” after the first hour, “2h+” and “2h-” after the second hour etc.
- At the end of the day, transfer 3 X 200µl aliquots of each of the samples in your ice bucket to the 96-well plate and measure using the fluorescent ELISA plate reader (Twinkle) downstairs.
Don't forget to upload the results from your group here