Microbiome DNA Isolation Kit

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

• An alternative elution buffer was used.

  It is recommended that the Elution Buffer B supplied with this kit be used for maximum DNA recovery.

• Lysis Additive A was not added to the lysate.

  Ensure that the provided Lysis Additive A is added to increase DNA yield. Also, an incubation can be performed at 65°C for 10 minutes after addition of the Lysis Additive A and prior to vortexing to maximize DNA recovery.

• Ethanol was not added to the lysate.

  Ensure that an equal amount of ethanol is 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:

• Elution is brown.

  Ensure that the Lysis Additive A is added. Also ensure Binding Solution I is added to the lysate and that it is incubated on ice for 10 minutes prior to spinning down the lysate. Avoid any contact with the pellet or surface residue when collecting the supernatant after the 5-minute spin during Sample Preparation.

• Lysis Additive A was not added to the lysate.

  Ensure that the provided Lysis Additive A is added to the lysate.

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

  Traces of salt from the binding step may remain in the sample if the column is not washed three times with the provided Binding Buffer B and Wash Solution A. 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.

• Binding Buffer I was not added to the lysate.

  Ensure that the Binding Buffer I is added to the lysate and that it is incubated on ice for 10 minutes prior to spinning down the lysate.

• PCR reaction conditions need to be optimized.

  Take steps to optimize the PCR conditions being used, including varying the amount of template, changing the source of Taq polymerase, looking into the primer design, and adjusting the annealing conditions.