Experimenting with aTc

From FreeBio

1. Inoculate overnight cultures in media with appropriate antibiotics; don't forget positive and negative controls.

2. Backdilute cultures to OD600 = 0.01 with LB + antibiotic; make enough cell solution to aliquot 2 ml for each concentration of aTc being tested. I find it helpful to have 2 ml per concentration plus 2 extra; when experimenting with 3 concentrations of aTc, for example, I would make a total of 8 ml of 0.1 OD600 solution. -- Connie

3. Aliquot 2 ml of solution into tubes.

4. Add 20 ul of aTc solution at appropriate concentration; add 20 ul DMF for negative control.

5. Incubate in 37 C shaker for 3 hours.

6. Put 500 ul of each culture into an eppendorf; spin down and aspirate.

7. Resuspend cell pellet in 15 ul of LB.

8. Place 1 ul of solution on slide.

9. Look at cells under microscope.

• Note: Adding aTc will cause cells to autofluoresce. Expect to see fluorescence under GFP and CFP filter, but not YFP.
• Current aTc stock found in the refridgerator is 100X = 2.5 mg/ml. Too much aTc tends to kill the cells; try 1.56 ug/ml or less.
• 50:50 serial dilutions are as follows: 25 ug/ml, 12.5 ug/ml, 6.25 ug/ml, 3.13 ug/ml, 1.56 ug/ml, 781 ng/ml, 391 ng/ml, 195 ng/ml