Description
ER0002
10X, 30 mL
Note: The procedure described below was performed
after plasmid DNA is produced in E. coli cells.
· Losses of up to 50% of the DNA are expected.
· Use of a DNA concentration above the
recommended 1 mg/ml reduces the efficiency of
the procedure.
1. Pipette 500 mL of the DNA solution into a sterile
microcentrifuge tube.
2. Add 50 mL of the 3 M sodium acetate solution to the
DNA sample.
3. Incubate on ice for 5 minutes.
4. Add 100 mL of cold Endotoxin Removal Solution.
5. Mix thoroughly and incubate on ice for 10 minutes.
The solution should be light blue and clear.
6. Incubate the tube at 37 °C for 20 to 30 minutes or
until the phases separate.
7. Spin for 5 minutes at 3000 x g in the
microcentrifuge. The upper phase is colorless and
clear, while the lower phase is blue.
8. Carefully transfer the upper phase containing the
DNA to a clean microcentrifuge tube.
9. Repeat steps 4 through 8 twice.
10. Add 0.6´ volume of 2-propanol. Mix by inversion at
room temperature and centrifuge at 15,000 x g for
30 minutes at 4 °C. Alternatively, add
2.5´ volumes of ethanol. Incubate overnight at -20
°C or 20 minutes at -70 °C and centrifuge at
15,000 x g for 30 minutes at 4 °C.
11. Carefully remove the supernatant
12. Wash the DNA pellet twice with cold 70% ethanol.
Remove the supernatant.
13. Air-dry the pellet.
14. Suspend the DNA in 100 mL of endotoxin free water
or TE buffer.
15. Determine DNA concentration and endotoxin levels
using endotoxin assay reagents and compare to
the starting material.
