GeneJET PCR Purification Kit

  • Add a 1:1 volume of Binding Buffer to completed PCR mixture (e.g. for every 100 µL of reaction mixture, add 100 µL of Binding Buffer). Mix thoroughly. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 µL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.
  • Optional: if the DNA fragment is ≤500 bp, add a 1:2 volume of 100% isopropanol (e.g., 100 µL of isopropanol should be added to 100 µL of PCR mixture combined with 100 µL of Binding Buffer). Mix thoroughly.
    Note: If PCR mixture contains primer-dimers, purification without isopropanol is recommended. However, the yield of the target DNA fragment will be lower.
  • Transfer up to 800 µL of the solution from step 1 (or optional step 2) to the GeneJET purification column. Centrifuge for 30-60 s. Discard the flow-through.
    Notes: 1. If the total volume exceeds 800 µL, the solution can be added to the column in stages. After the addition of 800 µL of solution, centrifuge the column for 30-60 s and discard flow-through. Repeat until the entire solution has been added to the column membrane. 2. Close the bag with GeneJET Purification Columns tightly after each use!
  • Add 700 µL of Wash Buffer (diluted with the ethanol as described on p. 3) to the GeneJET purification column.
  • Centrifuge for 30-60 s. Discard the flow-through and place the purification column back into the collection tube.
  • Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove any residual wash buffer.
    Note: This step is essential as the presence of residual ethanol in the DNA sample may inhibit subsequent reactions.
  • Transfer the GeneJET purification column to a clean 1.5 mL microcentrifuge tube (not included). Add 50 µL of Elution Buffer to the center of the GeneJET purification column membrane and centrifuge for 1 min.
    Note • For low DNA amounts the elution volumes can be reduced to increase DNA concentration. An elution volume between 20-50 µL does not significantly reduce the DNA yield. However, elution volumes less than 10 µL are not recommended. • If DNA fragment is >10 kb, prewarm Elution Buffer to 65°C before applying to column. • If the elution volume is 10 µL and DNA amount is ≥5 µg, incubate column for 1 min at room temperature before centrifugation.
  • Discard the GeneJET purification column and store the purified DNA at -20°C.
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