Revision as of 10:23, 27 November 2006 (view source)← Previous diff Current revision (05:13, 19 October 2007) (view source) (Removing all content from page) Line 1: Line 1: - Two parts: flow cytometry to determine distribution, and DNA concentration measurement to calibrate it. - - ===DNA Concentration=== - - # 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 Na2EDTA) 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. - - (Adapted from "Determination of DNA Content of Aquatic Bacteria by Flow Cytometry'' by - Button and Robertson, Applied and Environmental Microbiology, Apr 2001.) - - Controls: same procedure with no cells, vary the number of cells and see fluorescence variation - - 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=== - - # Grow ''M. smegmatis'' - # Stain with orange cell cycle stain from Invitrogen - - Get a pile of events.  The mean of this distribution should be the value measured above. - - ===Analysis=== - - 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?). - - 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.