Madhadron:PloidyMeasurement

Two parts: flow cytometry to determine distribution, and DNA concentration measurement to calibrate it.

DNA Concentration

1. Grow M. smegmatis
2. Take 100μL of culture, and do dilution plating to get population count.
3. Centrifuge for 20 minutes at 3,000rpm, resuspend to concentration ~10^9 cells/mL in STE buffer (0.1M NaCl, 50mM Tris HCl, 1mM Na₂EDTA) to ~10^7 cells/mL.
4. Add sodium dodecyl sulfate to 0.1%, incubate for 10 minutes at 60C
5. Add proteinase K (100μg/mL). Incubate at 37C for 30 minutes.
6. Add KCl to final concentration 40mM, incubate on ice for 30 min.
7. Centrifuge at 10,000g for 20 min at 5C, and discard pellet.
8. Stain supernatant with Hoechst 33258 at concentration 0.05 μg/mL.
9. Measure fluorescence at ex 350nm, em 450nm.

(Adapted from "Determination of DNA Content of Aquatic Bacteria by Flow Cytometry by Button and Robertson, Applied and Environmental Microbiology, Apr 2001.)

Compare number that comes out with Sigma's standard curve for calf thymus. Correct for mycobacterial GC/AT ratio as compared to calf.

Flow Cytometry

1. Grow M. smegmatis
2. Stain with orange cell cycle stain from Invitrogen

Get a pile of events. The mean of this distribution should be the value measured above.