Homogenization was incomplete - Depending on the type of stool, 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 5 minutes at maximum speed. Also, ensure that the maximum input of 200 mg of stool is not exceeded, as this may also cause incomplete homogenization.
An alternative elution buffer was used - It is recommended that the Elution Buffer supplied with this kit be used for maximum DNA recovery.
Lysis Additive was not added to the lysate - Ensure that the provided Lysis Additive is added to separate humic acid and increase DNA yield. Also, an incubation can be preformed at 65°C for 10 minutes after addition of the Lysis Additive 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 II - Ensure that 21 mL of 95 - 100% ethanol is added to the supplied Wash Solution II prior to use.
Why does the DNA not perform well in downstream applications?
Eluted DNA sample is brown - Ensure that the Lysis Additive is added. Also ensure Binding Solution 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 was not added to the lysate - Ensure that the provided Lysis Additive is added to the lysate.
DNA was not washed three times with the provided Wash Solutions - Traces of salt from the binding step may remain in the sample if the column is not washed three times with the provided Wash Solutions. 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 Solution was not added to the lysate - Ensure that the Binding Solution 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.
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