Team:SDU-Denmark/Protocols/Purification from gel

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=Protocol for purification of DNA from TAE and TBE agarose gel bands=
=Protocol for purification of DNA from TAE and TBE agarose gel bands=

Latest revision as of 10:03, 17 August 2009

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Contents

Protocol for purification of DNA from TAE and TBE agarose gel bands

Kit from GFX

Sample capture

  1. Weigh a DNase-free 1,5 ml microcentrifuge tube
  2. Excise band of interest from the gel and place in microcentrifuge tube
  3. Weigh microcentrifuge tube plus agarose gel band
  4. Calculate weight of agarose gel slice
  5. Add 10 ul Capture buffer type 2 for each 10 mg agarose gel slice
  6. Mix by inversion
  7. Place at 60 degrees until agarose is completely dissolved

Sample binding

  1. Add up to 600 ul Capture buffer-sample mix to assembled GFX MicroSpin columns and Collection tubes.
  2. Leave at room temperature for 60 sec.
  3. Centrifuge for 30 sec at 16000 g.
  4. Discard the flow through in the Collection tube and place the MicroSpin column in the Collection tube again.
  5. Repeat sample binding step until all sample is loaded onto the MicroSpin column.

Wash & dry

  1. Add 500 ul Wash buffer type 1
  2. Centrifuge for 30 sec at 16000 g.
  3. Discard flow through and keep Collection tube as above.
  4. Centrifuge again for 30 sec at 16000 g. More flow through will appear in the Collection tube. It is important to centrifuge this second time to get the sample completely dry. This step is not provided in the original protocol.
  5. Discard Collection tube and transfer MicroSpin column to a clean 1,5 ml DNase-free microcentrifuge tube.

Elution

  1. Add 10 – 50 ul Elution buffer type 4 or 6. We eluted with 10 ul in order to obtain a small volume and a high concentration of purified DNA. Only very big amounts of sample require higher elution volumes.
  2. Leave at room temperature for 60 sec.
  3. Centrifuge for 60 sec at 16000 g.
  4. Retain flow through and discard MicroSpin columns
  5. Store purified sample DNA at -20 degrees or proceed to cutting DNA or ligation.
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