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Wet Lab: Part Measurements and Characterisation
Characterisation of an inducible promoter:
- You have been provided with a 250ml conical flask containing 100ml of sterile LB media. Aseptically, add 5 ml of over-night culture with a 5ml sterile pipette to the flask. Add 100 µl of 50mg/ml Kanamycin (1000 X stock). Swirl to mix. Label this flask with your initials.
- Take out 1ml from the flask with a p1000 Gilson and transfer to a 1ml cuvette. One of the pair should measure the OD600 of this sample and record the readings in Table 2 on page 4. 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 the flask but transfer the aliquot to an Eppendorf tube labelled “0h” where the 0 represents the time in hours. Also, label the tube with your initials. Leave the tube in your ice bucket as this will be used for fluorescent measurements later on in the day.
- Put your 250ml conical flask in the 37°C shaking incubator.
- At 1hour intervals, repeat steps 2 & 3 until you have 6 OD600 readings and 12 or 13 (dependent on when you induce – after one or two hours) samples in your ice bucket for fluorescent measurements. *After 1-2 hours, when the OD600 ~ 0.4, take 50ml culture out and transfer it to a sterile 250ml flask. Induce ONE of your cultures by adding IPTG to the culture (you will be provided with the information about the IPTG concentration on the day). Swirl to mix. Then continue to take out samples from both cultures every hour after induction (for OD and fluorescence measurements).
- 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.
- While waiting for the OD to go up prepare:
Cell lysates from day 1.
- Dissolve the pellets in 250 µl PBS and transfer the tubes (using forceps) to the ice bucket containing dry ice. Freeze for 5 mins then thaw the tubes in the 25°C waterbath for 5 mins. Repeat freeze-thawing three times.
- Spin down the Eppendorf tubes in the microfuge at full speed for 10 mins. Make sure your tubes are balanced!
- After spinning, transfer the supernatants into new Eppendorf tubes and make sure not to transfer any cell debris. Leave the tubes on ice until you’re ready to do your fluorescent measurements.
- Transfer 200µl aliquots of each of the samples to the 96-well plate for the fluorescent reader. Remember to load one well with PBS as a negative control.
Don't forget to upload the results from your group here