MACHEREY-NAGEL – 03/2021, Rev. 05
14
RNA clean up XS
6
Appendix
6.1 Troubleshooting
Problem
Possible cause and suggestions
RNA is
degraded / no
RNA obtained
RNase contamination
• Create an RNase-free working environment. Wear gloves during
all steps of the procedure. Change gloves frequently. Use of
sterile, disposable polypropylene tubes is recommended. Keep
tubes closed whenever possible during the preparation. Glassware
should be oven-baked for at least 2 hours at 250 °C before use.
Poor RNA
quality or yield
Reagents not applied or restored properly
• Sample and reagents have not been mixed completely. Always
vortex vigorously after each reagent has been added.
• No ethanol has been added to Clean-up Buffer RCU. Binding of
RNA to the silica membrane is only effective in the presence of
ethanol. Adjust binding conditions by adding ethanol to Clean-up
Buffer RCU Concentrate as described in section 3.
• Store kit components at room temperature (18–25 °C). Storage
at lower temperatures may cause salt precipitation. If precipitation
occurs, incubate the bottle for several minutes at about 30–40 °C
and mix well until the precipitate is redissolved.
• Keep bottles tightly closed in order to prevent evaporation or
contamination.
Ionic strength and pH influence A
260
absorption as well as ratio A
260
/
A
280
• For adsorption measurement, use 5 mM Tris pH 8.5 as diluent.
Please see also:
- Manchester, K L. 1995. Value of A
260
/ A
280
ratios for measurement
of purity of nucleic acids. Biotechniques 19, 208–209.
- Wilfinger, W W, Mackey, K and Chomczyski, P. 1997. Effect of
pH and ionic strength on the spectrophotometric assessment of
nucleic acid purity. Biotechniques 22, 474–481.
Sample material
• Sample material not stored properly. Keep thawed samples on ice
before addition of Buffer RCU.
Contamination
of RNA with
genomic DNA
Sample material already contaminated with DNA
• Digest contaminating DNA in an RNA sample according to section
