Monique Mason, John Snider, Graham J. Threadgill, Ph.D., and Laura Pajak,
Ph.D.
Beckman Coulter, Inc.
| Problem |
Possible Cause |
Solution |
| Low or no yield: |
- Poor cell lysis: Over grown bacterial culture resulting
in incomplete resuspension of bacterial cell pellet or clogging
of Lysate Clearing plate.
|
- Limit cell density (as measured at A600) of 1.0 to 4.0
OD600 per well.
|
- Overgrowth of bacterial culture by nontransformed cells
or bacterial culture is too old.
|
- Inoculate antibiotic-containing media with single
bacterial colony from a fresh plate. Make sure antibiotic
is present in the media.
|
- No ethanol or not enough ethanol added to wash
solution.
|
- Prepare the wash solution according to the instructions
provided along with the kit.
|
- Inaccurate quantitation of plasmid DNA yield.
|
- Run the sample on an agarose gel along with
known quantity of DNA ladder, stain the gel with ethidium
bromide and quantitate by comparing the intensities.
|
- Incomplete cell lysis due to poor resuspension
of bacterial pellets.
|
- Thoroughly resuspend cell pellet. Recommended
centrifuge speed is 1,500 x g. Excessive
centrifugation will result in poor resuspension.
|
- Poor resuspension of bacterial pellets.
|
- Use the full resuspension cycle included in
the Wizard. Adjust the volume of the resuspension solution
to the default value (250 µl) or greater for large bacterial
pellets.
|
- Over growth of bacterial cultures, particularly
with strains containing Endonuclease I (EndA+) such as MC1061,
JM101, BL21.
|
- Grow cultures for a shorter time period, especially
when using TB broth (16 hrs). LB media 16-24 hrs. Saturation
conditions may cause cell lysis during culture resulting
in loss and degradation of plasmid DNA.
|
|
|
- Adjust vacuum pressure to 15” Hg with the vacuum
valve closed; reopen the valve before processing bacterial
plates.
|
|
| Filter plates not releasing from manifold
gaskets |
|
|
- Replace gaskets on manifold collar and spacer.
|
- Vacuum pressure too high.
|
- Adjust vacuum pressure to 15” Hg; alternatively,
increase the time in the “pause” steps after the vacuum
steps in the method to provide more time to allow for the
system to equilibrate to atmospheric pressure.
|
|
| Genomic DNA contamination |
- Too many mix cycles at resuspension step.
|
- Reduce number of mix cycles at resuspension step.
|
|
| Poor or no restriction digestion |
|
|
- Prevent ethanol contamination by allowing full 10 min
of drying and blotting of the filter plate.
|
- Too much DNA used in restriction digestion mix.
|
- Quantitate DNA accurately by agarose gel/ethidium
bromide electrophoresis.
|
- Concentration of restriction enzyme and length
of digestion is not optimal.
|
- Check the manufacture’s suggestions for restriction
enzymes including recommended restriction buffer, concentrations,
and suggested temperature. This is particularly important
when using multiple enzymes in a single digest reaction.
|
|
| A ratio of A260/A280
that is less than 1.7 |
|
|
- Overgrown cultures may result in carryover of protein
by overloading the SV 96 system. Repurify plasmid DNA.
|
- Spectrophotometric reading performed using water (<pH
6).
|
- Perform spec reading in neutral water or TE
(pH 7.0- 8.0). Some purified waters are acidic and cause
a increase in the A 280 reading, making it appear like protein
contamination.
|
|
| Poor or no results with automated fluorescent
DNA sequencing |
|
|
- Make sure there is no ethanol contamination by allowing
the full 10 min drying and affixing fresh blot paper for
the blotting step.
|
- Too much or too little DNA used in sequencing reaction.
|
- Quantitate DNA accurately by agarose gel/ethidium
bromide electrophoresis in addition to spectrophotometric
analysis.
|
- TE buffer used for plasmid DNA elution.
|
- Elute plasmid DNA only in nuclease free water
provided in the kit.
|
|
| Reagents are not delivered appropriately |
- Incorrect positioning of reagents when running method.
|
- Consult the instrument setup screen to ensure reagents
are placed in the appropriate location.
|
|
| Crashes occur when moving labware with
Gripper |
- Gripper fingers are worn, torn, or bent.
|
- Replace worn or torn finger pads, and bent fingers as
needed.
|
- The gripper or worksurface is inaccurately framed.
|
- Reframe the deck as necessary. Check framing
by moving a piece of labware on the deck before running
the method.
|
- Labware placed in the wrong position.
|
- Check instrument set up and make sure all labware
is in correct location.
|
- Improper labware definition.
|
- Review definition in the Labware Editor and
update if required. The labware can be reimported using
the Import file.
|
|
| Cannot move collar from the SPE ALP
to the Holder |
- Gripper inaccurately framed.
|
- Reframe gripper and/or adjust the squeeze/unsqueeze values
for the collar and collar spacer in the labware editor.
|
- Wrong 1x1 ALP placed adjacent to the Holder ALP.
|
- Place the 1x1 with the cut-out sides adjacent to the Holder
ALP. The cut-away sides allow the grippers to open to pick
up/release the collar on the Holder ALP.
|
|
| Vacuum bottle collapsing |
- Source vacuum is too high.
|
- Check that the dead end vacuum pressure is 15”Hg.
|
- Inappropriate bottle material.
|
- Check base of bottle for “PP” (Polypropylene)
designation. Replace with bottle made of correct material.
|
|
* 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.
