Team:IIT Bombay India/protocol



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Competent cell preparation of E.coli Strain MG1655(lacI deleted)

1.Take an overnight grown saturated E.coli cells in 5 ml LB and inoculate about 1/100 dilution in 50 ml culture. 2. Grow it at 37 degree Celsius till the O.D. reaches upto 0.5 to 0.8.

3. Transfer the culture to Cold autoclave round bottom centrifuge tube. Then keep them on ice for 10 minutes.

4.Spin at 5000 rpm for 10 minutes at 4 degree Celsius.

5.Decant the Supernatant.

6. keep the tube in upside down for 5 minutes on tissue paper pad to remove the residual medium.

7. Resuspend the pellet in 1/10 volume .1 M Cacl2.

8. Keep on ice for 40 minutes.

9. Spin at 5000 rpm for 10 minutes at 4 degree Celsius.

10. Discard the supernatant.

11. Resuspend the entire pellet in 1/25 volume of the .1 M cacl2.

12. Keep on ice for 60 minutes.

13. Add 15% glycerol (ice cold and autoclaved) to suspended cells.

14. Aliquotes in eppendorf tubes (ice cold) to a volume of 200ul each.

Transformation Of MG1655

1.	Thaw 200ul of competent cells on ice properly.

2.	Add around 100 nano gram of plasmid.

3.	Keep on ice for 30 minutes.

4.	Give heat shock at 42 degree Celsius for 90 sec to 120 minutes.

5.	keep on ice for 10 minutes.

6.	keep the tube at 37 degree Celsius for around half an hour.

7.	Spread around 100ul on the appropriate selection LB plates.

8.	keep the plates at 37 degree in incubator.

Growth curve

1.	Single colony was inoculated into 5ml LB with appropriate antibiotic.

2.	It was allowed to grow overnight.

3.	The culture O.D. was checked.

4.	It was added to 50 ml LB /Defined media to a volume of 1/100.

5.	Every one hour the O.D. was checked at 600 nm.

6.	O.D. was plotted against time on Excel sheet and Specific growth rate was calculated from the exponential phase.

Microscopy

1.Around 5ul of the culture was placed on a thin glass microscopic slide.

2.Mounted with coverslip.

3.Focused.

4.Exposed to blue laser.

5.Image was captured.

Fluorescence activated Cell sorting

1.Cells were grown for required time (for dynamics) or to a particular O.D. on M9 medium.

2.cells were pelleted at 4500 rpm for 10 minutes at room temperature.

3.Cells were washed twice in 1X PBS.

4.Cells were taken to a density of around 10 8 per ml.

5.The samples were exposed to blue laser for YFP and violet laser for CFP.

6. 5000 events were scored.

7.The Data was analysed and its statistical value was calculated with the help of BD FACSARIA software.

Beta galactosidase assay

1.	Grow 5ml LB cultures to mid-log phase.

2.	Centrifuge and resuspend cells in 5ml Z-buffer, then place on ice.

3.	Measure O.D.(600).

4.	Use straight, or dilute cell mix 10x or 20x (40 or 80ul brought to 0.8ml with Z-buffer).

5.	Using Pasteur pipet, add 1 drop of 0.1% SDS and 2 drops of chloroform to each tube.

6.	Vortex well for 15 seconds.

7.	Equilibrate at 30oC for 15 minutes.

8.	Add 160ul of 4mg/ml ONPG, and vortex well for 10 sec.

9.	Incubate at 30oC and begin timing.

10.	Remove after about 15-20 minutes (empirically determined by color).

11.	Quench reaction by adding 400 ul of 1M sodium carbonate.

12.	Spin down cell debris.

13.	Measure O.D.(420) and O.D.(550).

14.	Calculate Units using the following formula:

U= 1000 x [(OD420)-(1.75 x OD550)] / [(Time) x (Vol) x OD600]

Where Vol is volume of culture used in assay in mls, and Time is minutes at 30 degree C.

Z BUFFER (60mM Na2HPO4, 40mM NaH2PO4, 10mM KCl, 1mM MgSO4, 50mM 2-mercaptoethanol, pH 7.0)

To make 100ml:

•  6.0ml 1M Na2HPO4 stock solution •  4.0 ml NaH2PO4 stock solution

•  0.33 ml 3M KCL stock solution

•  1.0ml 100mM MgSO4 stock solution

•  350 ul 2-mercaptoethanol

Bring volume to 100 ml with distilled H2O. Do not autoclave!

ONPG

To make 10ml:

•  40 mg ONPG

•  10.0 ml 0.1 potassum phosphate buffer pH7.0

0.1M potassium phosphate buffer

To make 100ml:

•	Make solution A: 27.2 g KH2PO4 in 1 L water.

•	Make solution B: 34.8 g K2HPO4 in 1 L water.

Mix 39 ml Solution A and 61 ml Solution B and then add 100 ml of water.

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