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Plant RNA/DNA Purification Kit FAQ

Product No. E4788

  1. Why am I experiencing poor RNA recovery?
  2. Why is my RNA degraded?
  3. Why is my column clogged?
  4. Why does my RNA not perform well in downstream applications?
  5. Why is my yield of genomic DNA low?
  6. Why is my genomic DNA sheared?
  1. Why am I experiencing poor RNA recovery?

    Incomplete lysis of cells or tissue - Ensure that the homogenization step was done correctly with the appropriate amount of Lysis Solution for the amount of cells or tissue.

    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 also “Clogged Column” below.

    An alternative elution solution was used - It is recommended that the Nucleic Acid Elution Solution 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 Nucleic Acid Wash Solution - Ensure that 90 mL of 95 - 100% ethanol is added to the supplied Wash Solution prior to use.

    Low RNA content in cells or tissues used - Different tissues and cells have different RNA contents, and thus the expected yield of RNA will vary greatly from these different sources. Please check literature to determine the expected RNA content of your starting material. 
     
  2. Why is my RNA degraded?

    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 the 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°C for a few days. It is recommended that samples be stored at –70°C for longer term storage.

    Frozen tissues or cell pellets were allowed to thaw prior to RNA isolation - Do not allow frozen tissues to thaw prior to grinding with the mortar and pestle in order to ensure that the integrity of the RNA is not compromised.

    Tissue samples were frozen improperly - Samples should be flash-frozen in liquid nitrogen and transferred immediately to a -70°C freezer for long-term storage. 
     
  3. Why is my column clogged?

    Insufficient solubilization of cells or tissues - Ensure that the appropriate amount of lysis buffer was used for the amount of cells or tissue. Ensure that the lysate was incubated at 65°C for 10 minutes. Incubate the lysis solution for an extra 5 minutes to assist in lysis.

    Maximum number of cells or amount of tissue exceeds kit specifications - The optimal input of plant tissue is 50 mg or 5 x 106 plant cells. However, for most species, up to 100 mg of tissue may be processed

    Too much cell debris in the lysate supernatant - Ensure that most cell debris is removed in Step 1d.

    Centrifuge temperature too low - Ensure that the centrifuge remains at room temperature throughout the procedure. Temperatures below 20°C may cause precipitates to form that can cause the columns to clog. 
     
  4. Why does my RNA not perform well in downstream applications?

    RNA was not washed 3 times with the provided Nucleic Acid Wash Solution - Traces of salt from the binding step may remain in the sample if the column is not washed 2 times with Wash Solution. 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. 
     
  5. Why is my yield of genomic DNA low?

    Incomplete lysis of cells or tissue - Ensure that the appropriate amount of Lysis Solution was used for the amount of cells or tissue. Ensure that the lysate was incubated at 65oC for 10 minutes. Incubate the lysis solution for an extra 5 minutes to assist in lysis. Liquid nitrogen may be needed for lysis of challenging plant samples.

    The DNA elution is incomplete - Ensure that centrifugation at 14,000 x g for 1 minute is performed following the 2 minute centrifugation at 200 x g. Also, ensure that the entire volume of Nucleic Acid Elution Buffer passed through and is eluted from the column.
     
  6. Why is my genomic DNA sheared?

    Sample is old - Ensure that the sample is not too old, as old samples often yield only degraded DNA repeatedly frozen and thawed - Samples should not be repeatedly frozen and thawed, as this tends to increase the likelihood of isolating degraded DNA.