Biomedical Engineering Reference
In-Depth Information
Solution, Cell Lysis Solution, Neutralization Solution, and Column
Wash Solution are all found in the Promega kit. Perform all the
steps at room temperature, and label the tubes used during the
miniprep properly.
1. Centrifuge the 10 mL tube containing cells with DNA to be
isolated at 10,000×
g
for 5 min. Pour off supernatant and
remove excess media on a paper towel by inverting the tube.
2. Add 250 μL of Cell Resuspension Solution, and vortex or
pipette the solution until the pellet is completely resuspended.
Transfer to a sterile 1.5 mL microcentrifuge tube. For the
following steps, avoid vortexing and pipetting up and down,
not to induce double strand breaks in the DNA.
3. Add 250 μL of Cell Lysis Solution and mix by inverting the
tube four times. Incubate for 1-5 min, or until the cell suspen-
sion clears. Do not incubate for longer than 5 min.
4. Add 10 μL of Alkaline Protease Solution and mix by inverting
the tube five times. Incubate for 5 min (do not exceed this time).
5. Add 350 μL of Neutralization Solution and immediately mix
by inverting the tubes four times.
6. Centrifuge the bacterial lysate at 14,000 ×
g
for 10 min, using
a microcentrifuge.
7. Insert one Spin Column (found in the kit) into a 2 mL
Collection Tube (also in the kit), for each sample.
8. Transfer the lysate to the Spin Column, avoid transferring any
of the precipitate. If some of the precipitate is transferred, pour
the contents into a new sterile 1.5 mL microcentrifuge tube
and centrifuge again for 10 min. Transfer lysate.
9. Centrifuge for 1 min at maximum speed. Discard the flow
through and reinsert the Spin Column into the Collection Tube.
10. To wash, add 750 μL of Column Wash Solution to the Spin
Column.
11. Centrifuge for 1 min and discard flow through.
12. Repeat wash procedure, but use 250 μL of Column Wash
Solution instead.
13. Centrifuge for 2 min at maximum speed.
14. Carefully transfer the Spin Column to a sterile 1.5 mL micro-
centrifuge tube, so no Column Wash Solution is transferred.
Centrifuge for 1 min at maximum speed.
15. Transfer to a new sterile 1.5 mL microcentrifuge tube and
elute the plasmid DNA (now attached to the filter) by adding
100 μL Nuclease-Free Water to the Spin Column. Centrifuge
for 1 min at maximum speed.
16. Remove and discard the Spin Column. Close and label the
tube and store at −20 °C or below.
