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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