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Version 3.0 (August 1, 2003)
  • Drosophila genomic DNA purification using Biomek2000
    (Beckman Coulter)
    01/08/24 Misako Taniguchi

    Note: This protocol is optimaized for the use of dispensing robot, it should be applicable for manual purification with little modification. Those steps for which we use Biomek 2000 are indicated (Biomek).

    <Materials>

    • Racks with Collection tubes (QIAGEN: fits 96 well format)
    • Caps for collection tubes (QIAGEN)
    • Tungsten beads (QIAGEN)
    • Lysis buffer: 30mM Tris-Cl (pH 7.5), 30mM EDTA, 5% Tween-20, 0.5%TritonX-100, 4M GuHCl
    • Proteinase K stock solution: 20mg/ml Proteinase K, 50mM Tris-Cl (pH 7.5), 1mM CaCl2
    • 3M NaOAc (pH 5.6)
    • MultiScreen FB plate (MILLIPORE)
    • 70% ethanol
    • Reagent grade water (Milli Q)
    • TE (pH 8.0):10mM Tris-Cl (pH 8.0), 0.1mM EDTA
    • 96-well round bottom microtiter plate (IWAKI)
    • Adapter to connect a FB plate and a micro plate (MILLIPORE)
    • Easy Peel (ABgene)

    <Important notes before starting>

    • Set an incubator to 55oC for use (step 4)

      •
    • Set each tools at proper positions on Biomek2000 following the program 'GS-1 plate'

      • Place Wash8 and MP-200 at the A2 site
      • Place new tip racks of 200µl at the A6 site and B4.
      • Place a MILLIPORE Collar upon MILLIPORE Manifold at the A3 site.
    • Set distilled water at port 1 and 70% ethanol for at port 2 on Biomek2000

    <Methods>

    Lysis of flies

    1. Collect 20~30 flies into each collection tube. Seal a tube using a cap. After collecting the flies for one rack (96 tubes), store the rack at -80oC until next step.

      2.
    2. Prepare a working solution by gently mixing 4 ml ProteinaseK stock solution and 36 ml lysis buffer in a 50ml tube. Immediately, pour the mixture into a quarter reservoir and place on the A5 position of Biomek2000. Place a rack containing frozen flies prepared in step 1 at the A4 position of Biomek2000. Add 400µl of the mixture into the each collection tubes containing flies (Biomek). Seal the collection tubes using new caps. Ensure that the caps are properly sealed to avoid leakage during shaking.

    3. Mix thoroughly by shaking vigorously for 30 seconds. Spin for 10 seconds at 3000 rpm to collect any solution from the caps.

      4.
    4. Incubate at 55oC for 1 hour. Mix occasionally during incubation to disperse the samples, or place on a racking platform. Spin for 10 seconds at 3000 rpm to collect any solution from the caps.

    Adsorption to Millipore FB plate

    1. Pour 2 ml~ of 3M NaOAc (pH 5.6) into a quarter reservoir divided by length and then place the second site from the left at the A5 site on Biomek2000. Carefully remove the caps of the collection tubes and then place the rack at the A4 position on Biomek2000. Add 14 µl of 3M NaOAc (pH 5.6) to the each samples and mix them by pipetting for 3 times (Biomek).

      2.
    2. Place a new FB plate on MILLIPORE manifold at the A3. Take 150 µl of the sample solution from the each collection tubes, and transfer to the corresponding wells in the FB plate (Biomek).

      3.
    3. Adsorb genomic DNA to the membrane of the FB plate by applying vacuum to the FB plate (15 inches Hg) for 30 seconds (Biomek).

      4.
    4. Take 150 µl of the sample solution from the each collection tubes, and transfer to the corresponding wells in the FB plate again (Biomek).

      5.
    5. Adsorb genomic DNA to the membrane of the FB plate again: Vacuum from the bottom of the FB plate at 15 inches Hg for 1.5 minutes (Biomek). Removal of residual contaminants

      6.
    6. Wash the FB plate by 70 % ethanol for 2 times: Add 200 µl of 70 % ethanol to the each well of FB plate, and vacuum filtrate. Repeat this step 2 times (Biomek).

      7.
    7. Dry the FB plates by placing at 70 oC for 5 minutes and then heat 8 ml of TE to 70 oC.

    Elution of purified DNA

    1. Prepare a new 96-well round bottom micro plate. Put an adapter on the micro plate, and put the dried FB plate on them and place them at the A5 site on Biomek2000. Pour the preheated TE into a quarter reservoir and then place it at the third from the left at the A5 on Biomek2000.

      2.
    2. Add 70µl of the preheated TE to the each well of the FB plate (Biomek) and incubate for 5min at room temperature. Centrifuge at 2000 rpm for 10 minutes. Purified DNA is eluted from the FB plate into the 96-well round bottom micro plate. Seal the plate with Easy Peel (ABgene), close the lids of the plate and wrap with PARAFILM around the edge of the micro plate. DNA is stored at -80oC.
Copyrights (C) 2002 All rights reserved, Shigeo Hayashi (Riken Center for Developmental Biology) .
This database is based on the DYNACLUST system developed by DYNACOM Co., Ltd.