Poor DNA recovery could be due to one of the following factors:

• pH of the electrophoresis buffer was too high.

  Ensure that a fresh running buffer is used for electrophoresis. When the buffer is re-used, it often exhibits increased pH and may subsequently reduce yields.

• Gel slice was not completely melted in the Binding Buffer G.

  The gel slice should be incubated at 55°C until completely dissolved. The slice should be vortexed every 2 to 3 minutes to assist dissolving.

• Isopropanol was not added prior to binding.

  Ensure that 1 gel volume of Isopropanol is added to the melted gel slice prior to binding to the column.

• The appropriate amount of ethanol was not added to the Wash Solution A.

  The Wash Solution A has been specifically designed to contain the appropriate amount of components. Ensure that the Wash Solution A was prepared with the correct amount of 96-100% ethanol.

• Binding of DNA to the column was inefficient.

  Binding of the DNA is dependent on both pH and salt concentration. Ensure that an appropriate amount of Binding Buffer G was used for the weight of the gel slice.

• Binding Buffer G was not completely removed in the wash step.

  Traces of salt left on the column from the binding step may interfere with the elution of the DNA. Ensure that the column is washed with the Wash Solution A.

• Proper Elution Buffer B was not used.

  The provided Elution Buffer B has been optimized for high elution recoveries. If water or TE buffer is used instead, ensure the pH is around 8.

• Elution Buffer B was not placed directly onto the column bed.

  It is important that the Elution Buffer B be placed directly onto the column bed, as this helps to increase recovery by ensuring an even passing of the buffer through the column. Do not pipette the Elution Buffer B onto the side of the column.

Presence of impurities from the sample or the protocol can affect performance of DNA in downstream applications. This can happen when:

• Removal of Wash Solution A is incomplete.

  Ensure that the column is spun for 2 minutes during the wash step, in order to completely dry the column. Traces of Wash Solution A may remain in the eluted sample otherwise, and interfere with subsequent enzymatic reactions.

• DNA was not washed with the provided Wash Solution A.

  Traces of salt from the binding step may remain in the sample if the column is not washed with Wash Solution A. Salt may interfere with downstream applications, and thus must be washed from the column.