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280 Forde
2. Mix 100 μl of sample, 100 μl of the Picogreen working solution from step 1 above
and 1800 μl of TE buffer.
3. Allow the Picogreen to intercalate with the dsDNA for 30 s at room temperature.
4. Take a fluorescence reading at an excitation wavelength of 480 nm and an emission
wavelength of 520 nm.
5. Compare fluorescence readings for samples with those of a calibration curve
constructed using known concentrations of pDNA (supercoiled form) to determine
the concentration of dsDNA in the sample. Some dilution of the sample may be
required in order for the fluorescence reading to be within the linear range as
determined by the calibration curve.
3.6. Ethidium Bromide Agarose Gel Electrophoresis
1. Prepare the agarose gel by mixing 1× TAE buffer with 1.0% w/v agarose followed
by boiling to dissolve the agarose and homogenize the solution (see Note 13).
2. Allow the agarose solution to cool to below 60°C, then add ethidium bromide to
a concentration of 0.5 μg/ml of gel (see Note 14).
3. Pour the gel into a cast with a toothed comb to create the wells and allow to set.
4. Carefully remove the toothed comb from gel, then remove the gel from the cast
and place into the electrophoresis apparatus.
5. Pour 1× TAE buffer into the electrophoresis apparatus tank until the gel is just
covered.
6. Add 20% by volume sample loading buffer to each sample before loading into the
wells of the gel.
7. Run gels at 60 V for a minimum of 1horuntil the required resolution between
the bands had been obtained (see Note 15).
8. Photograph the gel using an appropriate gel documentation system (see Note 16).
4. Notes
1. A colony of DH5a Escherichia coli cells transformed with pTS were grown
overnight in 200 ml of inoculum media in a 2 l unbaffled shake flask. This cell
line was selected for the production of pDNA as it is relatively easy to transform
with pDNA, is well characterized and displays a high copy number (12). A high
copy number means that compared to other strains of bacteria, the number of
pDNA molecules that it produces per cell is high.
2. A 2 l working volume Applicon fermentation vessel linked to an Applicon ADI
1010 Bio Controller was used.
3. A cell culture temperature of 37°C was maintained via a water jacket and a pH
of 7 by use of 3 M NaOH and 3 M HCl additions.
4. The pTS plasmid confers ampicillin resistance to transformed E. coli.
5. DO was controlled to 30% of the maximum DO level by altering the agitation
speed (rpm) and compressed air or O 2 addition. The DO probe was calibrated by
running 4 l/min of pure O 2 through the system at an agitation speed of 800 rpm.