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| Two parts: flow cytometry to determine distribution, and DNA concentration measurement to calibrate it.
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| ===DNA Concentration===
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| # Grow ''M. smegmatis''
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| # Take 100μL of culture, and do dilution plating to get population count.
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| # Centrifuge for 20 minutes at 3,000rpm, resuspend to concentration ~10^9 cells/mL in STE buffer (0.1M NaCl, 50mM Tris HCl, 1mM Na<sub>2</sub>EDTA) to ~10^7 cells/mL.
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| # Add sodium dodecyl sulfate to 0.1%, incubate for 10 minutes at 60C
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| # Add proteinase K (100μg/mL). Incubate at 37C for 30 minutes.
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| # Add KCl to final concentration 40mM, incubate on ice for 30 min.
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| # Centrifuge at 10,000g for 20 min at 5C, and discard pellet.
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| # Stain supernatant with Hoechst 33258 at concentration 0.05 μg/mL.
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| # Measure fluorescence at ex 350nm, em 450nm.
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| (Adapted from "Determination of DNA Content of Aquatic Bacteria by Flow Cytometry'' by Button and Robertson, Applied and Environmental Microbiology, Apr 2001.)
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| Compare number that comes out with Sigma's standard curve for calf thymus. Correct for mycobacterial GC/AT ratio as compared to calf.
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| ===Flow Cytometry===
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| # Grow ''M. smegmatis''
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| # Stain with orange cell cycle stain from Invitrogen
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| Get a pile of events. The mean of this distribution should be the value measured above.
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