9
Spin Format
The Aurum plasmid mini kit can be used with any commercially available
microcentrifuge that can accommodate 1.5 ml and 2.0 ml tubes. All centrifugation
steps are performed at maximum speed ( 12,000 x g) under ambient conditions.
Please read the previous section "Guidelines for Using the Aurum Plasmid Mini
Kit" before proceeding.
1.
Transfer up to 12 OD•ml of plasmid-containing bacterial host to a 1.5–2.0 ml
capped microcentrifuge tube (not provided). Pellet the cells by centrifugation
for 1 min. Remove all supernatant by decanting or pipetting.
2.
Add 250 µl of resuspension solution and vortex or pipet up and down until
the cell pellet is completely resuspended.
3.
Add 250 µl of lysis solution and mix by inverting the capped tube briskly
6–8 times. DO NOT VORTEX OR SHAKE. The solution should become
viscous and slightly clear.
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. The supernatant or cleared
lysate contains the plasmid DNA.
6.
While centrifuging the lysate, insert a plasmid mini column into a 2 ml
capless wash tube (provided).
7.
By decanting or pipetting, transfer the cleared lysate from step 5 to the
plasmid mini column. Centrifuge for 1 min.
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.
Remove the plasmid mini column from the wash tube. Discard the filtrate
from the tube, and replace the column into the same wash tube. Add 750 µl
of wash solution and centrifuge for 1 min.
10. Discard the wash solution from the tube, and replace the column into the
same wash tube. Centrifuge for 1 additional minute 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 6).
