DNA Plant HTS 96 Kit

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Protocol :DNA extraction from plant material for use on a vacuummanifold1. Incubation of the plant material in Lysis Buffer PTransfer up to 50 mg plant material into the Qiawell plate and homogenize with Mixer Mill MM300 (Retsch) under liquid N 2 .Mix one bottle of Lysis Buffer P with one tube of Proteinase K and one bottle of RNase AAdd 400 µl Lysis Buffer P Mix (inclusive Proteinase K and RNase A) to each well of theQiawell plate .Incubate at 65 °C for 60 min, centrifuge for 10 min at 1.700 x g (4.000 rpm).2. PrefiltrationPlace the 1 ml Collection Plate in the base of the Invisorb ® 96 Vacuum Manifold and close themanifold with the top plate. Place the Prefilter Plate onto the rubber joint of the Invisorb ® 96Vacuum Manifold top plate.Transfer 350 µl Lysate carefully from Qiawell plate into the wells from the Prefilter Plate.Switch on vacuum source and apply vacuum up to the lysates are running through the filtercompletely.Discard the Prefilter Plate. Take out the Adapter and the 1 ml Collection Plate from the vacuummanifold base.3. Binding of the genomic DNA to the DNA Binding Plate CAdd 350 µl Binding Buffer P to each well of the 1 ml Collection Plate and mix the solutionvery well by pipetting several times up and down by using a multichannel pipet.Place the waste tray into the base of the Invisorb ® 96 Vacuum Manifold and close the manifoldwith the top plate. Place the DNA Binding Plate C onto the rubber joint of the Invisorb ® 96Vacuum Manifold top plate.Transfer the probes from 1 ml Collection Plate to the DNA Binding Plate C. Switch on vacuumsource and apply vacuum up to the lysates are running through the filter completely.4. First Washing of the DNA Binding Plate CAdd 500 µl Wash Buffer I to each well. Switch on vacuum source and apply vacuum until thetransfer of the wash buffer into the waste tray will be completed. Switch off vacuum and ventilatethe vacuum manifold.5. Second washing of the DNA-Binding Plate CAdd 700 µl Wash Buffer II to each well. Switch on vacuum source and apply vacuum until thetransfer of the wash buffer into the waste tray is complete. Switch off vacuum and ventilate thevacuum manifold.Repeat this washing step by adding again 700 µl Wash Buffer II to each well. Switch onvacuum source and apply vacuum until the transfer of the wash buffer is complete. Switch offvacuum and ventilate the vacuum manifold. To remove adherent fluid lift the DNA-Binding PlateC from the Invisorb ® 96 Vacuum Manifold top plate and strike the bottom side of the DNA-Binding Plate C on a stack of paper towels. Repeat several times until no further liquid isreleased onto the paper. Remove the manifold top plate from the base and discard the flowthroughfrom the waste tray.14Invisorb ® Plant DNA HTS 96 Kit /V and C 1010
6. Removing of ethanolPlace the waste tray inside the vacuum manifold and close the manifold with the top plate. Placethe DNA Binding Plate C onto the rubber joint of the Invisorb ® 96 Vacuum Manifold top plate.Switch on vacuum source and apply vacuum for at least 20 min for complete remove of allethanol. Switch off vacuum and ventilate the vacuum manifold. Take the DNA Binding Plate Cout of the manifold and place it on a clean paper towel. Remove the manifold top plate and thewaste tray from the base.7. Elution of the genomic DNAPlace a new 1.0 ml Collection Plate inside the base of the Invisorb ® 96 Vacuum Manifold andclose the manifold with the top plate. Place the DNA-Binding Plate C onto the rubber joint of theInvisorb ® 96 Vacuum Manifold top plate. Add 60 µl Elution Buffer D directly onto themembrane in each well and incubate for 5 min. Switch on vacuum source and apply vacuumfor at least 5 min until the transfer into the wells of the 1.0 ml Collection Plate is complete. Add60 µl Elution Buffer D again directly onto the membrane in each well. Switch on vacuumsource and apply vacuum for at least 5 min until the transfer into the wells of the 1.0 mlCollection Plate is complete.To remove adherent drops from the outlets of the DNA-Binding Plate C strike several times withthe flat hand on the top of the plate. Switch off vacuum and ventilate the vacuum manifold. Takethe DNA-Binding Plate C very carefully out of the manifold in order to avoid crosscontaminationswith adherent fluid. Discard the DNA-Binding Plate C. Remove the manifold topplate from the base and take the 1.0 ml Collection Plate with the eluted DNA out of the manifold.Cover the plate with the sealing foil.Note: Elution with prewarmed Elution Buffer D (up to 80 °C) will increase also the final yield.Invisorb ® 96 Vacuum ManifoldThe Invisorb ® 96 Vacuum Manifold contains acrylglas. To avoid any problems with thematerial please note the following instruction:1. Avoid a long incubation with ethanol or Binding Buffer P containing solutions.2. Clean up the manifold after using with water immediately and dry it.3. Always use the waste tray.15Invisorb ® Plant DNA HTS 96 Kit /V and C 1010
Page 2 and 3: ContentsKit content of Invisorb ®
Page 5 and 6: Safety InformationWhen working with
Page 7 and 8: Important points before starting a
Page 9 and 10: Scheme Invisorb ® DNA Plant HTS 96
Page 11 and 12: 3. Binding of the genomic DNA to th
Page 13: Scheme Invisorb ® DNA Plant HTS 96
Page 17 and 18: AppendixGeneral notes on handling D
Page 19 and 20: Invitek DistributorsAustraliaMedica

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