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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
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| - | Button and Robertson, Applied and Environmental Microbiology, Apr 2001.)
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| - | Controls: same procedure with no cells, vary the number of cells and see fluorescence variation
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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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| - | ===Analysis===
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| - | The DNA concentration gives a mean intensity $$\langle I_c \rangle$$ = a\langle n \rangle + b = f(s\langle n \rangle)$$, where $$f(\langle n \rangle)$$ we can find from Sigma's curves for DNA concentration vs. intensity, compensating for G/C content in mycobacteria, and $$s$$ is the weight of one chromosome. Let $$f_0 (\langle n \rangle)$$ be the curve from Sigma, $\gamma_{calf}$ be the GC/AT fraction in calf DNA and $\gamma_{myco}$ be that in mycobacteria. Then $f(\langle n \rangle) = $$ (FIXME! What does Hoechst bind to? Same thing as DAPI?).
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| - | The flow cytometry produces a set of intensity values. We let $$N(I)$$ be the number of chromosomes as a function of intensity, and assume it is linear, $$N(I) = cI+d$$. We find $$c$$ and $$d$$ by minimizing the three functions $$\langle N(I) \rangle - \langle n \rangle$$, $$\textrm{round}(N(I_m^i)) - N(I_m^i)$$, where I_m^i is the $i$th maximum of the density, which we assume must correspond to an integer.
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