Bio-Rad CHEF-DR II System - Manual (Page 20)

B. Bacteria and Yeast Cells:
Count five to ten of the 25 center squares, at 400x power, to get a representative sample
of your cell suspension. You should have approximately 25 to 75 cells per square. The cells
should be relatively free of clumps. Bacteria which naturally chain or grow in clusters are
relatively easy to count and do not have to be dispersed by chemical or enzymatic methods.
The Grams Crystal Violet aids in the visualization of bacteria.
For example: 300 bacteria in 5 squares = average of 60 bacteria/square x 25 (squares) x
20 (dilution factor, yeast use 100 for dilution factor, or whatever dilution factor you prefer to
use) x 10
4 = 3 x 10 8 bacteria per ml. So for 5 ml of plugs you need 5 ml x 5 x 10 8 cells final
concentration ÷ 3 x 10 8 cells/ml concentration = 0.83 ml of cell suspension is required.
3.9 Estimation of Agarose Plug DNA Concentration:
Two pieces of information are needed to determine DNA concentration:
1. The size in base pairs of the genome. This information is readily available for most organ-
isms, otherwise a best guess is necessary. We use 6 x 10 9 for mammalian, 4.5 x 10 6 for
Escherichia coli and 1.5 x 10 7 for Saccharomyces cerevisiae in the following examples.
2. You need to determine the number of genomes per cell. For example, for stationary growth
phase in yeast or bacterial cells or confluent growth in tissue culture cells, assume one
genome per cell. However, for exponential phase growing cells there is more than one
genome per cell. Make a best guess or assume one per cell which will give the minimum
concentration of DNA. In the below examples we use a value of 1.2 genome equivalents
(20%) for mammalian cells, 2.5 genome equivalents for bacteria, and 2 genome equiva-
lents for yeast.
217
Equations for Estimation of DNA Concentration in Agarose Plugs
(A)
(B)
(C)
(D)
17
(Genome Size bp)(660 g/mole)
= grams DNA/cell
6.02 x 10 23 bp/mole
(grams DNA/cell)(cell/ml) = (grams DNA/ml)(1x10 6 µg/g) = µg DNA/ml
(µg DNA/ml)(genome equivalents (Section 3.9.2))
≅
(µg DNA/ml)
µg DNA/ml ≅ µg DNA/plug ≅
µg DNA/lane
10 plugs/ml 2 lanes/plug (Section 3.7.3)