Team:TU Dresden/Notebook/Protocols/HiYieldGelPCRDNAExtractionKit


HiYield Gel/PCR DNA Extraction Kit

  1. Gel Dissociation: Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice (USE only TAE buffer for gel formation). Transfer up to 300 mg of the gel slice to a 1.5 mL microcentrifuge tube. Add 500 μL of DF Buffer to the sample and mix by vortex.

    Incubate at 55-60 °C for 10-15 minutes to ensure the gel slice has been completely dissolved. During incubation, invert the tube every 2-3 minutes. Cool the dissolved sample mixture to room temperature.

  2. DNA Binding: Place the DF Column in a 2 mL Collection Tube. Transfer 800 μL of the sample mixture to the DF Column. Centrifuge at 14-16,000 g for 30 seconds. Discard the flow-through and place the DF Column back in the 2 mL Collection Tube. Repeat the step if the mixture is more than 800 μL.

  3. Wash: Add 300 μL of W1 Buffer into the DF Column. Centrifuge at 14-16,000 g for 30 seconds then discard the flow-through. Place the DF Column back in the 2 mL Collection Tube. Add 600 μL of Wash Buffer (make sure ethanol was added) into the DF Column. Let stand for 1 minute at room temperature.

    Centrifuge at 14-16,000 g for 30 seconds then discard the flow-through. Place the DF Column back in the 2 mL Collection Tube. Centrifuge at 14-16,000 g for 3 minutes to dry the column matrix.

  4. DNA Elution: Transfer the dried DF column to a new 1.5 mL microcentrifuge tube. Add 20-50 μL of Elution Buffer or TE into the center of the column matrix. Let stand for at least 2 minutes to ensure the Elution Buffer is completely absorbed. Centrifuge for 2 minutes at 14-16,000 g to elute the purified DNA.