eLabNotebook > Nucleic Acid Prep & Purification > Plasmid DNA Purification > QIAGEN QIAprep
Biomek® 2000


A Fully Automated Method for Purification of Plasmid DNA on the Biomek® 2000 Laboratory Workstation Utilizing the QIAGEN QIAprep* 96 Turbo Miniprep Kit

Troy Cook, Ryan McCrae, and Paul W. Diaz, Ph.D.
Beckman Coulter, Inc.

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Problem Possible Cause Solution
Low or no yield:
  • Low yields may be caused by a number of different factors. To find the source of the problem analyze fraction saved form each step in the procedure on an agarose gel. A small amount of the cleared lysate and the entire flow-through can be precipitated by adding 0.7 volumes isopropanol and centrifuging at maximum speed (1,000 x g or 13,000 rpm) for 30 minutes. The entire wash flow-through can be precipitated by adding 0.1 volumes of 3M sodium acetate, pH 5.0 and 0.7 volumes of isopropanol.
No DNA in the cleared lysate before loading
  • Plasmid did not propagate.
  • Check that the conditions for optimal growth for bacterial cultures were met.
  • Lysate prepared incorrectly.
  • Check storage conditions and age of buffers.
  • Buffer P2 precipitated.
  • Redissolve by warming to 37° C.
  • Cell resuspension incomplete
  • Pelleted cells should be completely resuspended in buffer P1. Do not add buffer P2 until an even resuspension is achieved.
DNA is found in the flow-through of cleared lysate
  • QIAprep membrane is overloaded.
  • If rich media such as TB or 2x YT are used, culture volumes must be reduced. It may be necessary to adjust LB culture volume if the plasmid and host strains show extremely high copy number or growth rates.
  • RNase A digestion omitted.
  • Ensure that RNase A is added to buffer P1 before use.
  • RNase A digestion insufficient.
  • Reduce culture volume if necessary. If buffer P1 containing RNase A is more than 6 months old, add additional RNase A.
DNA is found in the wash flow-through
  • Ethanol omitted from the buffer.
  • Repeat procedure with correctly prepared wash buffer (buffer PE).
Little or no DNA in the eluate
  • Elution buffer incorrect.
  • DNA is eluted only in the presence of low salt buffer, e.g., Buffer EB (10mM Tris-Cl, pH 8.5) or H2O. Elution efficiency is dependant on pH. The maximum efficiency is achieved between pH 7.0 and 8.5. When using water for elution, make sure the pH value is within this range.
Low DNA quality 
DNA does not perform well
  • Eluate salt concentration too high.
  • Ensure that two wash steps are completed prior to elution.
  • Nuclease contamination.
  • When using endA+ host strains such as HB101 and its derivatives, the JM series or any wild type strain, ensure that the wash step with Buffer PB is performed.
  • Eluate contains residual ethanol.
  • Ensure that the 10 minute vacuum and plate blot are performed prior to elution.
Low DNA quality 
RNA in the eluate
  • RNase A digestion omitted.
  • Ensure that RNase A is added to buffer P1 before use.
  • RNase A digestion insufficient.
  • Reduce culture volume if necessary. If buffer P1 containing RNase A is more than 6 months old, add additional RNase A.
Low DNA quality 
Genomic DNA in the eluate
  • Buffer P2 added incorrectly.
  • The lysate must be handled adding buffer P2 to prevent shearing. Reduce culture volume if lysate is too viscous for gentile mixing.
  • Buffer N3 added incorrectly.
  • Mix buffer N3 immediately upon addition.
  • Lysis too long.
  • Lysis step must not exceed 5 minutes.
  • Culture overgrown.
  • Overgrown cultures contain lysed cells and degraded DNA. Do not grow cultures for more than 12-16 hours.
Pipette Functions
  • Reagents are not delivered to the correct sample well locations; Samples are not transferred to the correct location on the filter plate.
  • Two global patterns are used for aspirating and dispensing in each pipette transfer. One pattern is for each pipette function that involves the sample source labware and the other pattern is for pipette functions that involve the vacuum filtration labware. When processing less than 96 samples, the location of the samples in the source plate may differ from the location of samples at their destination in the filtration labware. Since the patterns are global they only need to be set once in the pattern editor and they are changed in each pipette transfer function that uses them in every method in the current lab book. The patterns can be set without opening the method in method editor.
Running the Method
  • Work surface setup.
  • The worksurface is setup so that when processing samples, possible contamination to downstream items, tips / labware, due to fly-over is minimized.
  • Why can't I start the method with the vacuum filtration assembled?
  • Placement of the QIAprep plate in the vacuum manifold is critical. The wells must align with the wells of the QIAfilter plate in the Vacuum collar. Pacing the QIAprep plate in the QIAwell Holder registers the plate in the correct position and the gripper will then place it in the vacuum manifold in the correct position to catch the filtrate in the proper wells. The QIAwell holder is actually a modular reservoir holder that is place in a labware holder with QIAprep plate in it. The QIAwell holder is placed on the worksurface in method edit as an edited labware holder.
Vacuum System
  • Filter plate sticking to vacuum collar.
  • Clean gasket and lightly oil with silicone if required.

    Replace old or worn gasket, Black Neoprene recommended. Vacuum may still be present, set system pause to allow enough time for equalization.
  • Collar sticking to Vacuum Manifold.
  • Dirty manifold O-ring, Clean with alcohol.

    Vacuum may still be present, set system pause to allow enough time for equalization.
  • Gripper fingers catching on plate, collar or manifold.
  • Worksurface and / or gripper Y alignment out of adjustment. Critically align Biomek and gripper.

    Grip width out of adjustment. Perform gripper alignment.

    Improper device / labware definition. Review definition in the source lab book and update if required.
  • Vacuum bottle collapsing
  • Source vacuum greater than 20" Hg. Adjust dead end vacuum to read 20" Hg at pump.

    Regulator malfunctions or out of adjustment, with source vacuum at 20", adjust regulator to 15" at dead end.

    Bottle Fatigue. Once collapsed it will fail again, replace bottle!
  • Wash 8 is not aspirating well
  • Possible leak in the aspirate plumbing.

    Vacuum pinch valve on wash unit not opening.
  • Care and cleaning.
  • It is recommended that you flush and dry the wash 8 tool after each use.

    Use the automatic wash method "Wash & Clean" to flush the wash 8 tool and the vacuum manifold and vacuum valve unit simultaneously.

    Clean manifold and collar gaskets with Alcohol periodically.

* 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.

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