media when 10–12 OD•ml of bacteria are processed per column, although
smaller amounts of culture may also be processed.
To determine the density of a bacterial culture (OD
600
), combine 50 µl of
bacterial culture with 950 µl growth medium (1:20 dilution). Use the growth
medium as a blank and take the spectrophotometric reading at
l
= 600 nm.
Multiply this figure by 20 to calculate the bacterial concentration. Depending
upon the OD
600
value, a specific volume of the culture will be selected to
provide an optimum amount of bacteria for processing. To calculate the
volume of bacterial culture required for plasmid purification, use the following
equation:
(OD
600
of undiluted culture)* x (culture volume in ml) = #OD•ml
For example, 12 OD•ml of bacteria would require 2 ml of an undiluted
culture with an OD
600
= 6.
• The protocol is designed to process up to 12 OD•ml of bacterial host.
*OD
600
is equivalent to approximately 8 x 10
8
cells/ml
Vacuum Guidelines:
• The recommended operating range is -20 to -23 inches of mercury (" Hg).
Do not exceed -25" Hg when performing this protocol. A vacuum regulator
is required to establish the appropriate vacuum pressure (Figure 2).
Table 1. Pressure unit conversions
To convert from inches of mercury (" Hg) to:
Multiply by:
millimeters of mercury or torr (mm Hg, torr)
25.4
millibar (mbar)
33.85
atmospheres (atm)
0.03342
pounds per square inch (psi)
0.4912
kilopascals (kPa)
3.385
4
