eLabNotebook > Nucleic Acid Prep & Purification > Genomic DNA Purification > Promega SV 96 gDNA Purification Biomek® FX


High-Throughput Genomic DNA Isolation from Cell Cultures and Mouse Tails on the Biomek® FX Liquid Handling System using Promega's Wizard* SV 96 Genomic DNA Purification System

Hobert Wai, Ph.D.**, Matthew Cu**, Dana P. Campbell**, and Keith Roby**
Beckman Coulter, Inc.
**Indianapolis, IN and ***Fullerton, CA

Terri Grunst and Daniel Kephart
Promega Corporation – www.promega.com

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.

Click here to download a printable PDF version of the following:

Problem Possible Cause Solution
Deck mapping and setup difficulties.
  • Calling up the wrong deck.
    Not enough ALP positions.
  • Refer to Beckman Coulter Application Information Bulletin T-1953, “Deck Mapping on the Biomek® FX.”
Low DNA yield
(i.e., low A 260).
  • Sample contained too few cells.
  • Obtain fresh new cell samples.

  • Check to see if cell count is within the recommended range.
  • Tissue lysate is stored at -20 or -70°C.
  • For optimal yield, purify the DNA as soon as the lysate is prepared. Generally, if the lysate has been frozen it may have a decreased amount of genomic DNA.
  • Sample subjected to many freeze-thaw cycles.
  • Freezing and thawing samples repeatedly could result in DNA degradation. Use fresh samples whenever possible.
  • Tissue culture cells were low in genomic DNA.
  • Genomic DNA yield varies
    depending on the number of cells used for the isolation. Increase the starting amount processed to a maximum of 20 mg of tissue or 5 X 106 tissue culture cells.
  • Sample did not receive any or enough Wizard SV Lysis Buffer.
  • Process lysates at 55°C
    incubation. If samples have
    cooled, it may be difficult to purify due to viscosity. Incubate at 55°C for 1 hour and continue with the
    purification.
  • Lysed cells or lysates were not re-suspended completely.
  • Increase number of sample mixes to ensure re-suspension before transferring to the DNA filter plate.
  • No ethanol or incorrect volume of ethanol added to the Wizard SV Wash Solution.
  • Prepare the solutions as instructed in Promega’s Technical Bulletin No. 303 Sections III.A and IV.A
    before beginning the procedure.
  • Low vacuum pressure.
  • Check vacuum pump for correct pressure (15 inches of Hg).

  • Check vacuum lines and seals for leakage.
Inaccurate elution volume.
  • There is not enough nuclease-free water.
  • Make sure to have enough elution water.

  • Allow 10 mL overage when adding to the reservoir.
  • The aspirating height is not adjusted to the volume of the nuclease-free water.
  • Adjust the aspirating height
    according to the amount of
    nuclease-free water.
  • Vacuum pump is not working properly.
  • Check to see that vacuum pump is set to the required pressure. A vacuum pressure of >15 inches of Hg is recommended.

  • Make sure that vacuum hoses are in good conditions (i.e., no holes or deterioration) and correctly connected—see the Biomek® FX User’s Manual #719452.

  • Check the manifold and collar gaskets for leakage.
Clogged column.
  • Lysate was too viscous to pipette easily.
  • Dilute lysate with Wizard SV Lysis Buffer until it becomes easy to pipette. Then apply the entire lysate to the column.
  • Too much tissue sample was used in the lysate preparation.
  • The maximum recommended weight of mouse tail tissue used for lysate preparation is 20 mg.
  • Too many cells were being processed.
  • The maximum recommended amount of tissue culture cells being processed on the column
    membrane is 5 X 106 cells.
  • Tissue lysates were too viscous when cooled.
  • Process lysates at 55°C incubation. If samples have cooled, it may be difficult to purify due to viscosity.
    Incubate at 55°C for 1 hour and continue with the purification.
  • Differential vacuum pressure in filter plate.
  • 1. Manually place Vacuum Assist film from Whatman, Cat. # 7705-0112, on the filter plate during vacuum steps to ensure even vacuum pull in all 96 columns.
Uneven reagent distribution.
  • Uneven multi-channel pipetting head.
  • Inspect the levelness of the
    multi-channel pipetting head by lowering the pod via manual control to touch a 1X1 ALP on the deck. Uneven contact of the head to the ALP signifies a tilted head. Contact a service engineer to relevel the pipetting head before operating the Biomek® FX.
  • Clogged mandrel or tips.
  • Need to refurbish head and
    inspect tips for blocks.
  • Loading partially filled tip boxes.
  • Only full tip boxes can be used. Partially filled tip boxes creates uneven insertion force on the
    multi-channel pipetting head, which may cause damage and pipette inaccuracy to the multichannel pipetting head.
Reagents are not
delivered correctly.
  • Incorrect positioning of reagents.
  • Consult the instrument setup screen to ensure reagents are placed in the appropriate location.

  • Check to make sure that lysis buffer, wash solution, and nuclease-free water are added to the appropriate labware and placed in the correct deck position.
  • Reagents were not delivered to the desired position in wells.
  • Frame deck properly.
RNA contamination.
  • RNase A was not added to the tissue lysate digestion solution.
  • Add 2 uL of RNase A solution to final eluate and incubate at room temperature for 10 minutes.
  • RNA was co-purified with genomic DNA from tissue culture cells.
  • Add 2 uL of RNase A solution to final eluate and incubate at room
    temperature for 10 minutes.
Contamination of wells.
  • Outside sources introduced during sample preparation.
  • Make sure that Biomek® FX and reagents are free of contaminants.

* 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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