IGEM:IMPERIAL/2009/M3/Assays/IPTG effects2: Difference between revisions
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====Inoculation of cells==== | ====Inoculation of cells==== | ||
* Inoculate single colonies of E. coli cells into 15ml falcon tubes containing 5 ml of the pre-warmed (37°C) supplemented M9 medium with | * Inoculate single colonies of E. coli cells into 15ml falcon tubes containing 5 ml of the pre-warmed (37°C) supplemented M9 medium with spectinomycin (20 ug/ml) | ||
* Grow the cultures for O/N with spinning at 70 rpm. | * Grow the cultures for O/N with spinning at 70 rpm. |
Revision as of 13:06, 26 August 2009
Dry lab test experiment: IPTG effect on growth
Aims
- Measure growth rates at 28 degrees Celsius on minimal growth media so that subsequent testing timings etc. can be streamlined
- Determine the effect of IPTG toxicity on growth w/o any protein production complications
Assay
Normal cells (without any constructs) will be grown on M9 media supplemented with glucose and secondary carbon source until OD=0.7.
IPTG will then be added, and the OD of the cells will be followed over time.
Equipment
- Spectrophotometer
- 600nm absorbance filter
- 15ml falcon tubes
- cuvettes
Reagents
Media
Total volume: 120ml
M9 minimal media – 11.28g/L of 5x powder
MgSO4 – 0.4g/L
Glycerol (5.0g per L)= 54.39uM 0.5%
Glucose (0.5g per L) = 2.78mM 0.05% per 100ml
kanamycin (20 ug/ml) = 1mg/500ml
Cultures
E.coli cells (TOP-10 strain)
Others
IPTG (0.1 g)
Protocol
Thurs 11AM:
Things needed
- Minimal Media constituents
M9 Minimal Media Preparation:
- Measure out the following reagents and dissolve them in 1000ml of sterile H20:
M9 Minimal Media- 11.28g/L of 5x powder
Glucose (0.5g per L) = 2.78mM 0.05%
Glycerol (5.0g per L)= 54.39uM 0.5%
Maltose (5.0g per L)= 14.6mM 0.5% per 100ml
Autoclave the M9 media to ensure sterility.
MgSO4 - 0.4g/L powder
To maintain sterility, MgSO4 solution should be filter sterilised (Minisart® 0.20µm syringe filter) and added after the other chemicals had been dissolved, mixed and autoclaved
Thurs 1PM:
Things needed
- Minimal Media constituents
- 15ml falcon tubes
Inoculation of cells
- Inoculate single colonies of E. coli cells into 15ml falcon tubes containing 5 ml of the pre-warmed (37°C) supplemented M9 medium with spectinomycin (20 ug/ml)
- Grow the cultures for O/N with spinning at 70 rpm.
Will take 20hrs based on TOP10-DH5a in Jason Kelley Paper
Fri 9AM:
Things needed
- IPTG 0.1g
- Minimal Media constituents
- 15ml falcon tubes
1) Growing up of cultures
- Dilute the cultures which are at a high cell density 1:20 into 2 conical flasks of 50 ml of fresh media each and grow the cultures at 28°C in an incubator.
2) IPTG solution Preparation:
- 0.1g of IPTG dissolved in 0.4ml of dH2O (filter sterilize) to get 1M IPTG
3) Labelling of plates
- Label 2 conical flasks setups:
Flask 1: 0 uM IPTG
Flask 2: 1 mM IPTG
Fri 12PM:
Things needed
- 1M IPTG solution
- Spectrophotometer
- Cuvettes
Monitering of OD
- After 3 hours, 1ml of solution from each flask is transferred to a cuvette.
- The OD is obtained by measuring the absorbance at 600nm using the spectrophotometer. The OD should be around 0.7. If not, wait longer.
- After the OD reaches 0.7, add 49ul of 1M IPTG to flask 2 and mix well.
- Repeat the absorbance measurement every hour for both flasks during mid-exponential growth for 6 hours
- Determine background absorbance by measuring blank with only media. This should be subtracted from subsequent absorbance readings.