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Plasmid DNA Purification 281
Initially, compressed air at 4 l/min was supplied to the vessel. The gas was
changed from compressed air to pure O 2 when the system failed to maintain a
DO level of 30% at the maximum agitation speed of 800 rpm.
6. It is good practice to check the OD 600 nm reading of the inoculum to ensure
that the transformed colony has indeed grown. A low cell concentration in the
inoculum results in a long lag phase of cell growth and is an inefficient use of
time and resources. The preparation of more than one inoculum will assist in the
success of the bacterial fermentation protocol.
7. The length of a fermentation run is dependent upon the system used: cell line,
pDNA, inoculum used, temperature, medium, pH, DO and so on. Generally, for
the system described above, a lag time of approximately 2 h was witnessed before
the system entered the exponential growth phase. Smaller inoculum volumes
extend the initial lag time. OD 600 nm readings of up to 23.9 were obtained after 24
h of fermentation; however, fermentation runs of this length are not necessarily
required as the maximum volumetric yield of supercoiled pDNA can be obtained
as soon as 10 h after inoculum addition.
8. The length of time for this step is dependent upon the cell concentration. Cell
lysis is normally complete after 5–10 min. Leaving the solution too long may
result in the yield, and/or supercoiled nature of the pDNA being compromised
as the pDNA is then unable to renature upon neutralization.
9. This step removes the precipitated floc formed after neutralization. The majority
of host cell-derived contaminants (gDNA, proteins and cell debris) precipitate to
form fragile salt aggregated flocs after neutralization. The advantages of alkaline
lysis are that it has a high capacity for cell-derived contaminant removal and
is fully scalable. Care must be taken to prevent high shear during lysis as this
results in a lower yield of supercoiled pDNA and fragmentation of gDNA.
10. Protein and reduced glutathione will be present in this eluted product. The pDNA
and free fusion protein elute at different rates, so this enables some removal of free
fusion protein from the fractions that contain the highest concentration of pDNA.
EDTA is a very potent zinc-chelating agent. EDTA treatment (2 mM) leads to
irreversible denaturation and aggregation of the zinc-binding domain that cannot
be restored by addition of an excess of zinc (13). If further removal of protein and
reduced glutathione from the pDNA is required, incubate the elution fractions
in a solution containing 2 mM EDTA, then separate the denatured protein and
reduced glutathione from the pDNA using size exclusion chromatography or a
buffer exchange method.
11. The binding capacity of the affinity adsorbent can be affected by the accumulation
of precipitate, denatured or nonspecifically bound proteins that are not
removed by the relatively mild high and low pH adsorbent regeneration buffers.
Precipitated and/or denatured substances can be removed by washing with 2
column volumes of 6 M guanidine hydrochloride followed by washing with 5
column volumes of PBS. Hydrophobically bound substances can be removed by
washing with 4 column volumes of 70% v/v ethanol followed by washing with
5 column volumes of PBS (14).