Soil DNA Isolation Plus Kit

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

• Homogenization was incomplete.

  Depending on the type of soil, further vortexing with the flat bed vortex or bead beater equipment may be required. However, it is not recommended to increase the vortex time to longer than 10 minutes at maximum speed.

• Lysis Additive A was not added to the lysate.

  Ensure that the provided Lysis Additive A is added to separate humic acid and increase DNA yield.

• Lysis Buffer QP and Ethanol were not added to the lysate.

  Ensure that 400 µL of Lysis Buffer QP and 550 µL of 96-100% ethanol are added to the lysate before binding to the column.

• Ethanol was not added to the Wash Solution A.

  Ensure that 42 mL of 96-100% ethanol is added to the supplied Wash Solution A prior to use.

If the DNA does not perform well in downstream applications, it may be due to one or more of the following:

• Eluted DNA sample is brown.

  The elution contains high humic acids. Ensure that the OSR Solution was added to the clean lysate. Also, ensure the column was washed with Binding Buffer B.

• DNA was not washed with the provided Binding Buffer B and Wash Solution A.

  Traces of humic acids or salt from the binding step may remain in the sample if the column is not washed with the provided Binding Buffer B and Wash Solution A. Humic acids and salt may interfere with downstream applications, and thus must be washed from the column.

• Ethanol carryover.

  Ensure that the dry spin under the Column Wash procedure is performed in order to remove traces of ethanol prior to elution. Ethanol is known to interfere with many downstream applications.

• PCR reaction conditions need to be optimized.

  Take steps to optimize the PCR conditions being used, including varying the amount of template (10 ng to 50 ng for 20 µL of PCR reaction is recommended), changing the source of Taq polymerase, looking into the primer design, and adjusting the annealing conditions.