eLabNotebook > Nucleic Acid Prep & Purification > Plasmid DNA Purification > Promega SV 96 Twelve-Plate Biomek® FX


High Throughput Generation of Quality Plasmid DNA on the Biomek® FX using the Wizard* SV 96 Plasmid DNA Purification System from Beckman Coulter, Inc.

Jason Fawcett, Monique Mason, and Laura Pajak, Ph.D.
Beckman Coulter, Inc.

For additional information try our Maintenance and Troubleshooting section or post a question to our Biomek User Forum.  New members can subscribe to our User Forum here.

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 pellet.
  • 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 (250µl) 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.
  • Low vacuum pressure.
  • Adjust vacuum pressure to 15” Hg with the vacuum valve closed; reopen the valve before processing bacterial plates.
  • Vacuum valve not working or configured properly.
  • Check that the vacuum valve has been set to the correct com port and you can hear a ‘clicking’ noise as the valve opens and closes.
Filter plates not releasing from manifold gaskets
  • Worn, older 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.
  • The Labware Jog move is missing in the method steps.
  • Make sure that any method modifications did not remove the jog step. Re-run the Wizard to restore the original method.
Genomic DNA contamination
  • Too many mix cycles at resuspension step.
  • Reduce number of mix cycles at resuspension step.
Poor or no restriction digestion
  • Ethanol contamination.
  • 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
  • Protein contamination.
  • 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 an increase in the A 280 reading, making it appear like protein contamination.
  • Samples are contaminated.
  • Make sure that the water source has enough volume to accommodate the entire run. Otherwise the tips will not be washed properly which may result in sample contamination. It may be easier to have a continuous supply by connecting a line directly to a water source to ensure an adequate supply.
Poor or no results with automated fluorescent DNA sequencing
  • Ethanol contamination.
  • 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.
  • Circulating reservoir pump not turned on.
  • The circulating reservoir pump is independently controlled and does not automatically turn on. Before starting a run make sure that the pump is on and that the liquid in the reservoirs has had enough time to reach their operating volume.
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.
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.
Method Errors
  • Stacker Carousels cannot be detected.
  • Make sure that the stacker carousels are connected to the correct com port and are configured properly in the hardware setup utility.
  • Stacker Carousels have not been configured properly for the correct hotel types.
  • Select the appropriate hotel stack in the hardware setup. For this method all the stacker carousel hotels are stack 10’s.
  • There is no device associated with the SC1 and SC2 deck positions.
  • In the deck editor make sure that the SC1 and SC2 positions have a stacker device associated with that position. For position SC1 the device should be Stacker1 and for position SC2 the device should be Stacker2.
  • The software cannot detect the FX.
  • Make sure the Biomek FX is connected to the correct com port on the computer and then select that com port in the hardware setup.
  • No device controller installed.
  • The wash pumps will not work if the device controller is not installed properly. Check that there is a device controller installed in the hardware setup utility and the corresponding wash pumps are also installed.
  • No device associated with wash stations.
  • In the deck editor make sure that each wash position has a wash pump associated with the position.
  • Wash pump on wrong line on the device controller.
  • Connect the wash pumps to the appropriate line on the device controller and maker sure the wash pumps are correctly configured in the hardware setup utility.
  • Beckman pump installed in simulation when the Wizard was installed.
  • Reinstall the Wizard and select install pump with real hardware.
  • The additional roving height is too high.
  • Adjust the additional roving height in the pod editor to 0.55cm. This will allow enough space for the Biomek to move things around the deck without giving a Z-max error.
  • The min safe height for the stacker carousels is too high.
  • In the deck editor adjust the min safe height for the stacker carousels until the error is removed. Typically, decreasing the min safe height will clear the error.
  • Attempting to frame the holder position on the 2x1 ALP.
  • The 2x1 ALP only needs the SPE position framed. The holder position does not need framing.
  • Beckman pumps not installed on correct com ports.
  • Under the device setup menu make sure that each Beckman pump is connected to the correct com port.
  • Stacker carousels cannot initialize because they have labware blocking the sensors.
  • Remove the top pieces of labware from Hotels A and C. Turn the stacker carousel on and off. Once they have re-initialized replace the removed labware.

* 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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