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DNeasy Blood and Tissue Collection Procedure

Equipment and reagents to be supplied by user

  • Pipets and pipet tips
  • Vortexer
  • Ethanol
  • Microcentrifuge tubes (1.5 ml or 2.0 ml)
  • Microcentrifuge with rotor for 1.5 ml and 2 ml tubes
  • Thermomixer, shaking water bath, or rocking platform for heating at 56 °C

Safety Equipment

  • Lab coat
  • Safety goggles
  • Disposable gloves

Maximum amount of starting material

  • Mouse Tail: 0.6-1.2 cm

Protocol: Purification of DNA from Animal Tissues (Including Mouse Tails)

Important Points

  • Centrifugation steps should be carried out at 15-25 °C in a microcentrifuge
  • Vortexing should be performed by pulse-vortexing for 5-10s

Before Starting

  • Buffer ATL and AL may form precipitates during storage. If necessary, warm to 56 °C until the precipitates have dissolved.
  • Buffer AW1 and AW2 are supplied as concentrates. Add the appropriate amount of ethanol (96-100%) as indicated on the bottle to obtain a working solution.
  • Preheat a thermomixer, shaking water bath, or rocking platform to 56 oC for use in step 2.

Procedure

  1. Place two mouse tail sections, 04-0.6 cm in length, into a microcentrifuge tube. Add 180 µl Buffer ATL. Earmark the animal appropriately.
  2. Add 20 µl proteinase K. Mix thoroughly by vortexing, and incubate at 56 oC until the tissue is completely lysed. Vortex occasionally during incubation to disperse the sample, or place in a thermomixer, shaking water bath, or on a rocking platform.
    • Lysis time is 6-8 hours for rodent tails. They can be lysed overnight for convenience this will not adversely affect DNA collection.
    • If the lysate appears gelatinous refer to "Troubleshooting Guide". Note: it may appear viscous and this is normal, but a very gelatinous consistency will clog the DNeasy Mini Spin Column.
  3. Vortex for 15s. Add 200 µl Buffer AL to the sample, an mix thoroughly by vortexing. Then add 200 µl ethanol (96-100%), and mix again thoroughly by vortexing.
    • The sample, Buffer AL, and ethanol must be mixed immediately by vortexing or pipetting to yield a homogeneous solution. Buffer AL and ethanol can be premixed and added together in one step to save time when processing multiple samples.
    • If a white precipitate forms on addition of Buffer AL and ethanol vigorously shake or vortex the preparation.
  4. Pipet the mixture from step 3 (including any precipitate) into the DNeasy Mini spin column placed in a 2 ml collection tube (provided). Centrifuge at ¿ 6000xg (8000 rpm) for 1 min. Discard flow-through and collection tube.
  5. Place the DNeasy Mini spin column in a new 2ml collection tube (provided), add 500 µl Buffer AW1, and centrifuge for 1 min at ¿ 6000xg (8000 rpm). Discard flow-through and collection tube.
  6. Place the DNeasy Mini spin column in a new 2 ml collection tube (provided), add 500 µl Buffer AW2, and centrifuge for 3 min at 20,000 x g (14,000 rpm) to dry the DNeasy membrane. Discard flow-through and collection tube.
    • It is important to dry the DNeas Mini spin column, since residual ethanol may interfere with subsequent reactions.
    • Following centrifugation, remove the DNeasy Mini spin column carefully so that the column does not come into contact with the flow-through, since this will result in carryover of ethanol. If carryover of ethanol occurs, empty the collection tube, then reuse it in another centrifugation for 1 min at 20,000 x g (14,000 rpm).
  7. Place the DNeasy Mini spin column in a clean 1.5 ml or 2 ml microcentrifuge tube (not provided), and pipet 200 µl Buffer AE directly onto the DNeasy membrane. Incubate at room temperature for 1 min, and then centrifuge for 1 min at ¿ 6000xg (8000 rpm) to elute.
  8. Recommended: For maximum DNA yield, repeat elution once as described in step 7.
    • This step leads to increased overall DNA yield
    • A new microcentrifuge tube can be used for the second elution step to prevent dilution of the first eluate. Alternatively, to combine the eluates, the microcentrifuge tube from step 7 can be reused for the second elution step.
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