E.Z.N.A.® Gel Extraction Kit (V-spin)

How can I improve recovery and get higher A260/A230 ratios using the E.Z.N.A. Gel Extraction Kit?

Few recommendations:

1. Use running buffers made fresh. (Reusing the same running buffer messes with the pH of binding and, in fact, inhibits DNA binding to the column).
2. Double the binding buffer (XP2) to dissolve the gel pieces if the gel pieces are hard to dissolve. You would have to run the DNA/agarose solution through the column more than once in this case. Instead of heating in step 5, pipette the solution up and down until the agarose is completely dissolved. This results in a higher recovery percentage with larger fragments in our experiments.
3. After the Gel/Binding Buffer mixture has completely dissolved, monitor the color. If the color of the mixture becomes orange or red, add 5 µL 5M sodium acetate (pH 5.2) to bring the pH down. After this adjustment, the color of the Gel/Binding Buffer mixture should be light yellow. It needs to be yellow for good binding onto the column (most times, reusing running buffers messes up the binding pH and hence recovery).
4. Heat the elution buffer to 70 °C and add to the column directly. Incubate on the column for 5 minutes and perform a second elution step. That is, pipette the first eluate back on the column and spin it down.

Low A260/A230 ratios alludes to salt carryover (guanidine in the binding buffer is not completely washed off during wash steps). Typically increasing the wash steps improves this ratio – so instead of 2 SPW wash steps, do 3.

Can the E.Z.N.A. Gel Extraction Kit be used for PCR clean-up as well?

The E.Z.N.A. Gel Extraction Kit (D2500) can be used for both PCR clean-up and gel extraction.

What is the binding capacity of the column in D2500?

Our HiBind® DNA Mini Columns can bind DNA between 100 bp – 10 kbp of DNA and the binding capacity of the column is 100 µg.