Equipment
Omega plasmid extraction kit.
Protocol
1. Isolate a single colony from a freshly streaked
selective plate, and inoculate a culture of 1-5 mL LB medium
containing the appropriate selective antibiotic. Incubate
for~12-with a volume of at least 4 times the volume of the
culture. It is strongly recommended that an endA negative strain
of E. coli be used for routine plasmid isolation. Examples of
such strains include DH5a® and JM109®.
2. Centrifuge at 10,000 x g for 1 minute at room
temperature.
3. Decant or aspirate and discard the culture media.
4. Add 250 uL Solution I/RNase A. Vortex or pipet up and
down to mix thoroughly. Complete resuspension of cell pellet is
vital for obtaining good yields.
Note: RNase A must be added to Solution I before use.
Please see the instructions in the Preparing Reagents section
on Page 6.
5. Transfer suspension into a new 1.5 mL microcentrifuge
tube.
6. Add 250 uL Solution Il. Invert and gently rotate the
tube several times to obtain a clear lysate. A 2-3 minutes
incubation may be necessary.
Note: Avoid vigorous mixing as this will shear
chromosomal DNA and lower plasmid purity. Do not allow the
lysis reaction to proceed more than 5 minutes. Store Solution
Il tightly capped when not in use to avoid acidification from
CO, in the air.
7. Add 350 uL Solution Ill. Immediately invert several
times until a flocculent white -precipitate forms.
Note: It is vital that the solution is mixed thoroughly
and immediately after the addition of Solution Ⅲ to avoid
localized precipitation.
8. Centrifuge at maximum speed (≥13,000 x g) for 10
minutes. A compact white pellet will form. Promptly proceed to
the next step.
9. Prepare the vacuum manifold according to
manufacturer's instructions.
10. Connect the HiBind® DNA Mini Column to the vacuum
manifold.
Optional Protocol for Column Equilibration
1. Add 100 uL3M NaOH to the HiBind® DNA Mini Column.
2. Turn on the vacuum source to draw the NaOH through the
column.
3. Turn off the vacuum.
11. Transfer the cleared supernatant from Step 8 by
CAREFULLY aspirating it into the HiBind® DNA Mini Column. Be
careful not to disturb the pellet and that no cellular debris is
transferred to the HiBind® DNA Mini Column.
12. Turn on the vacuum source to draw the sample through
the column.
13. Turn off the vacuum.
14. Add 500 uL HBC Buffer.
Note: HBC Buffer must be diluted with isopropanol
before use. Please see Page 6 for instructions.
15. Turn on the vacuum source to draw the buffer through
the column.
16. Turn off the vacuum.
17. Add 700 uL DNA Wash Buffer.
Note: DNA Wash Buffer must be diluted with 100% ethanol
prior to use. Please see Page 6 for instructions.
18. Turn on the vacuum source to draw the buffer through
the column.
19. Turn off the vacuum.
20. Repeat Steps 17-19 for a second DNA Wash Buffer wash
step.
21. Transfer the HiBind® DNA Mini Column to a 2 mL
Collection Tube.
22. Centrifuge the empty HiBind® DNA Mini Column for 2
minutes at maximum speed to dry the column matrix.
Note: It is important to dry the HiBind® DNA Mini
Column matrix before elution. Residual ethanol may interfere
with downstream applications.
23. Transfer the HiBind® DNA Mini Column to a clean 1.5
mL microcentrifuge tube.
24. Add 30-100 uL Elution Buffer or sterile deionized
water directly to the center of the column membrane.
25. Let sit at room temperature for 1 minute.
26. Centrifuge at maximum speed for 1 minute.
Note: This represents approximately 70% of bound DNA.
An optional second elution will yield any residual DNA, though
at a lower concentration.
27. Store DNA at -20℃.
