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

• 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 H was used for the volume of the DNA input.

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

  The Wash Solution K has been specifically designed to contain the appropriate amount of components. Ensure that the Wash Solution K was prepared using the correct amount of ethanol.

• Binding Buffer H 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 K.

• Proper Elution Buffer was not used.

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

• Elution Buffer L was not placed directly onto the resin.

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

• Insufficient washing of the column.

  Ensure the column is washed thoroughly to remove any impurities that may affect DNA recovery. Proper washing with Wash Solution K is essential.

• Sample viscosity is too high.

  High viscosity can impede the flow through the column. Dilute the sample if necessary to reduce viscosity and improve binding efficiency.

• Incorrect storage of reagents.

  Ensure all reagents are stored according to the manufacturer's instructions. Improper storage can affect the performance of the buffers and overall DNA recovery.

This could be due to insufficient washing of resin with bound DNA. Traces of salt from the binding step may remain in the sample if the column is not properly washed with the Wash Solution K. Ensure that the column is spun for 2 minutes during the washing step. Salt may interfere with downstream applications, and thus must be washed from the column.

The CleanAll DNA/RNA Clean-Up and Concentration Micro Kit is designed to reduce endotoxin levels to below 0.1 EU/μg of DNA. However, higher endotoxin levels can be observed due to one or a combination of the following factors:

• Proper Elution Buffer was not used.

  The provided Elution Buffer L has been optimized for endotoxin-free recoveries. If endotoxin-free water is used for the elution, ensure that the pH is between 7 and 8.

• A different Elution Buffer was used.

  The provided Elution Buffer L has been optimized for endotoxin-free recoveries. The endotoxin-free properties of the eluted DNA will be compromised if another elution buffer is used. If a different elution buffer other than the one provided is used, the buffer should also be checked for endotoxin levels.

• The endotoxin levels of the input were extremely high.

  If the initial input DNA had extremely high endotoxin levels, the levels may not be completely reduced to 0.1 EU/μg of DNA or less. In this case, the eluted DNA could be applied to a second column and the procedure repeated in order to further reduce the endotoxin levels.

Poor RNA recovery could be due to one or a combination of the following factors:

• Column has become clogged.

  Do not exceed the recommended amounts of starting materials. The amount of starting material may need to be decreased if the column shows clogging below the recommended levels. See FAQ related to “Clogged Column” below.

• An alternative elution solution was used.

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

• Ethanol was not added to the lysate.

  Ensure that the appropriate amount of ethanol is added to the lysate before binding to the column.

• Ethanol was not added to the Wash Solution K.

  Ensure that 54 mL of 96-100% ethanol is added to the supplied Wash Solution K prior to use.

Column clogging can result from one or a combination of the following factors:

• High amounts of RNA as input.

  Ensure that no more than 50 µg of RNA are used as input.

• High amounts of genomic DNA present in sample.

  The lysate may be passed through a 25 gauge needle attached to a syringe 5-10 times in order to shear the genomic DNA prior to loading onto the column.

• Centrifuge temperature too low.

  Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 15°C may cause precipitates to form that can cause the columns to clog.

Eluted RNA can be degraded due to the following factors:

• RNase contamination.

  RNases may be introduced during the use of the kit. Ensure proper procedures are followed when working with RNA. Please refer to “Working with RNA” at the beginning of this user guide.

• Procedure not performed quickly enough.

  In order to maintain the integrity of the RNA, it is important that the procedure be performed quickly.

• Improper storage of the purified RNA.

  For short term storage RNA samples may be stored at –20℃ for a few days. It is recommended that samples be stored at –70℃ for longer term storage.

If purified RNA does not perform well in downstream applications, it can be due to the following reasons:

• RNA was not washed twice with the provided Wash Solution K.

  Traces of salt from the binding step may remain in the sample if the column is not washed twice with Wash Solution K. 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.

Using large amounts of starting material can cause DNA or genomic DNA contamination in the RNA fraction. Perform RNAse-free DNase I digestion on the RNA sample before clean-up according to the protocol provided in Appendix A.

Elution buffer L is an EDTA-free buffer.