7
Note:
The neutralization solution should be added within 5 min after lysis.
4.
Add 350 µl of neutralization solution and mix by inverting the capped tube
briskly 6–8 times. DO NOT VORTEX OR SHAKE. A visible precipitate should
form.
5.
Centrifuge the neutralized lysate for 5 min. A compact white debris pellet will
form along the side or at the bottom of the tube.
6.
While centrifuging the lysate, attach a plasmid mini column to the luer fitting of
the column adaptor plate on the Aurum vacuum manifold or to a compatible
vacuum manifold. The vacuum source should be turned off, and the vacuum
regulator should be completely open.
7.
By decanting or pipetting, transfer the cleared lysate from step 5 to the
plasmid mini column. Turn the vacuum on and adjust to -20 to -23" Hg by
closing the vacuum regulator. Continue to apply vacuum until all of the lysate
has passed through the column. Open the vacuum regulator until the gauge
indicates 0" Hg.
8.
The wash solution is supplied as a 5x concentrate. Add 4 volumes (100 ml) of
95–100% ethanol or reagent-grade (denatured) ethanol before initial use.
9.
Add 750 µl of wash solution to the column and close the vacuum regulator
dial until the gauge indicates -20 to -23" Hg. Continue to apply the vacuum
until all wash solution has passed through the columns. Open the vacuum
regulator until the gauge indicates 0" Hg.
10. Transfer the plasmid mini column to a 2 ml capless wash tube (provided) and
centrifuge for 1 min to remove residual wash solution.
11. Transfer the plasmid mini column to a 1.5–2.0 ml capped microcentrifuge
tube (not provided). Add 50 µl of elution solution onto the membrane stack
at the base of the column and allow 1 min for the solution to saturate the
membranes. Centrifuge for 1 min to elute the plasmid.
12. Discard the mini column and store the eluted DNA at 4ºC.
A protocol overview is available (see Figure 5).
