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6
Appendix
6.1
Troubleshooting
Problem
Possible cause and suggestions
RNase contamination
•
RNA is
degraded / no
RNA obtained
Reagents not applied or restored properly
•
•
•
•
Poor RNA
quality or yield
Ionic strength and pH influence A
A
•
Sample material
•
Sample material already contaminated with DNA
Contamination
of RNA with
•
genomic DNA
14
RNA clean up XS
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.
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.
280
For adsorption measurement, use 5 mM Tris pH 8.5 as diluent.
Please see also:
- Manchester, K L. 1995. Value of A
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 not stored properly. Keep thawed samples on ice
before addition of Buffer RCU.
Digest contaminating DNA in an RNA sample according to section
5.2.
MACHEREY-NAGEL – 03/2021, Rev. 05
absorption as well as ratio A
260
/ A
ratios for measurement
260
280
/
260
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