| Problem |
Possible Cause |
Solution |
| Incomplete resuspension of cells (cell pellet still visible) |
- Cells stored too long at –20°C
|
- Cells should only be stored 2–4 weeks, maximum, at –20°C. Shake an additional 5 minutes.
|
|
| Incomplete resuspension of cells (cell pellet still visible) |
- Too many cells, biomass greater than 6.0 O.D.600
|
- Do not attempt to process more than 6.0 O.D.600 of total cell mass per well.
|
|
| Incomplete resuspension of cells (cell pellet still visible) |
- Cells not thawed completely
|
- When using frozen cells, ensure
that they are completely thawed.
Allow the cells to sit at room
temperature at least 15 minutes.
|
|
| Turbidity observed in cleared lysate |
- Too long at neutralization step
|
- Do Not shake longer than 4
minutes. The lysate will remain
cloudy and decreased plasmid
DNA yield will result.
|
|
| Turbidity observed in cleared lysate |
|
- Make sure frozen cells are
completely thawed.
Increase lysis time from 3 to 5
minutes.
|
|
| Turbidity observed in cleared lysate |
- Too many cells, biomass greater than 6.0 O.D.600
|
- Increase lysis time from 3 to 5 minutes. Do not attempt to process more than 6.0 O.D.600
per well.
|
|
| Compact pellet does not form at magnet corner, and complete removal of cleared lysate is not possible. |
- Too many cells, biomass greater than 6.0 O.D.600
|
- Do not attempt to process more than 6.0 O.D.600 of total cell mass.
|
|
| Compact pellet does not form at magnet corner, and complete removal of cleared lysate is not possible. |
- Growth medium is interfering with protocol
|
- We recommend Terrific broth or CIRCLEGROW* medium for this protocol.
|
|
| Compact pellet does not form at magnet corner, and complete removal of cleared lysate is not possible. |
- Magnetic flux may be insufficient
|
- Use only the Greiner 96-well plates or plates of the same
design.
|
|
| Downstream applications are problematic |
|
- Thorough washing is required to
remove salts that might interfere
with downstream applications.
Insure that the magnetic particles
are thoroughly suspended in the
well by the action of the shaker or
by pipetting at each wash step.
|
|
| Downstream applications are problematic |
|
- Ensure that all the wash
is removed from the well.
If alcohol is spilled while shaking,
lessen the amplitude.
|
|
| Low DNA yield |
|
- Thoroughly resuspend the
particles after adding the
Nuclease-Free Water.
|
|
| Low DNA yield |
- Insufficient mixing of binding resin and lysate
|
- The binding resin and lysate must be thoroughly mixed to ensure maximum binding.
|
|
| Particle carryover to final elution plate |
|
- Reduce the aspiration rate.
|
|
| Particle carryover to final elution plate |
- Particles adhering to tip walls
|
- If re-using tips or using fixed tips,
rinse them to remove adherent
particles.
|
|
| Particle carryover to final elution plate |
- Particles adhering to tip walls
|
- Add a second magnetic step to
remove particles.
|
|
* All trademarks are the property of their respective owners. Where applicable,
the PCR process is covered by patents owned by Roche Molecular Systems,
Inc., and F. Hoffman-LaRoche, Ltd.