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Plasmid DNA Purification 279
5. Decant the clarified cell lysate (pDNA containing supernatant) into a clean
container. Prepare cell lysate on the day it is to be used.
3.3. Expanded Bed Adsorption Plasmid DNA Purification
1. Load a chromatography column via gravity settling with the adsorbent prepared
as described in Chapter 9 (see Subheading 3.1.).
2. Equilibrate the column with at least 10 settled bed column volumes of PBS buffer
using upward flow to expand the column. Expand the bed to twice its settled bed
height. In a 1-cm diameter column, a flow rate of approximately 150 cm/h is
required to expand the bed to twice its settled bed height.
3. Using upward flow, load GST–ZnF-containing lysate into the column. Some
column expansion should be expected due to the higher density and viscosity of
the feed. To prevent loss of adsorbent through the top of the column, the flow
may need to be reduced or the position of the top column frit adjusted.
4. Wash the column with at least 5 settled bed column volumes of PBS buffer. Ensure
that the OD 280 nm of the column outlet stream returns to base-line levels.
5. Still in expanded mode, load the pDNA containing clarified cell lysate into the
column, followed by 5 settled bed column volumes of PBS buffer to wash the
column.
6. Reverse the flow to downward flow and lower the top adaptor. Continue washing
with PBS until the OD 280 nm of the column outlet stream returns to base-line levels.
7. Elute the GST–ZnF–pTS complex in packed bed mode with elution buffer and
collect the elution fractions for off-line analysis via ethidium bromide agarose gel
electrophoresis and Picogreen assays (see Note 10).
3.4. Affinity Adsorbent Regeneration
1. After elution is complete, signified by a stable OD 280 nm , reverse the flow to the
upward flow direction and expand the column to twice its settled bed height using
high pH adsorbent regeneration buffer. Pump 5 settled bed column volumes of
high pH adsorbent regeneration buffer through the column.
2. Still in expanded bed mode, pump 5 settled bed column volumes of low pH
adsorbent regeneration buffer through the column.
3. Repeat steps 1 and 2 a further two times or until no more material is eluted from
the affinity adsorbent, which is shown by a stable OD 280 nm for the column outlet
stream (see Note 11).
4. Wash the column with 5 bed volumes of PBS.
5. For long-term storage (i.e., several weeks or more), wash the column with 5 bed
volumes of adsorbent storage buffer and store at 4°C.
3.5. Picogreen Fluorescence Assay
1. Mix one part of Picrogreen as supplied with 199 parts of TE buffer to produce a
Picogreen working solution (see Note 12).