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Wet Lab: Part Measurements and Characterisation
Characterisation of a Stationary/Log phase detector
- You have been provided with 2 x 250ml conical flasks containing 100ml of sterile LB media. Aseptically, add 5 ml of over-night culture 995 with a 5ml sterile pipette to one of the flasks. Then add 100 µl of 50mg/ml Ampicillin (1000 X stock). Swirl to mix. Label this flask with your initials and “995”.
- Repeat step 1 by aseptically adding 5 ml of over-night culture 996 to the other flask. Then add 100µl of 50mg/ml Ampicillin (1000 X stock). Swirl to mix. Label this flask with your initials and “996”.
- Take out 1ml from each flask with a p1000 Gilson and transfer to 1ml cuvettes. The sample from the 995 flask should be labelled 995” and the sample from the 996 flask should be labelled “996”. One of the pair should measure the OD600 of both samples and record the readings in Table 3 on page 5. 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 “0h995” and “0h996” where the 0h 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.
- Put your 2 x 250ml conical flasks in the 37°C shaking incubator.
- At 1 hour intervals, repeat steps 3 & 4 until you have 6 OD600 readings and 6 “995” and 6 “996” samples in your ice bucket for fluorescent measurements. For the fluorescence samples, label your Eppendorf tubes “1h 995” and “1h 995” after the first hour, “2h 995” and “2h 996” 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 (do not use the outer wells of the plate!) and measure using the fluorescent ELISA plate reader (Twinkle) downstairs.
- Leave your 2 x 250ml flasks shaking in the 37°C shaking incubator overnight.