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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''
 
−  # Take 100μL of culture, and do dilution plating to get population count.
 
−  # 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.
 
−  # Add sodium dodecyl sulfate to 0.1%, incubate for 10 minutes at 60C
 
−  # Add proteinase K (100μg/mL). Incubate at 37C for 30 minutes.
 
−  # Add KCl to final concentration 40mM, incubate on ice for 30 min.
 
−  # Centrifuge at 10,000g for 20 min at 5C, and discard pellet.
 
−  # Stain supernatant with Hoechst 33258 at concentration 0.05 μg/mL.
 
−  # 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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−  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''
 
−  # 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.
 