Protocols; Rna Clean Up And Concentration Of Rna - Macherey-Nagel NucleoSpin RNA Clean-up XS User Manual

5

Protocols

5.1

RNA clean up and concentration of RNA

Before starting the preparation:
• Check if Buffer RCU and Buffer RA3 were prepared according to section 3.
1 Sample preparation
Provide up to 300 µL sample containing up to 90 µg
RNA – such as prepurified RNA (e.g., phenol purified) or
RNA from reaction mixtures (e.g., labelling reactions) –
in a microcentrifuge tube (not provided).
For appropriate sample amounts see section 2.2.
Note: Fill up RNA samples smaller than 100 µL with
RNase-free water to 100 µL. RNA samples from
100–200 µL should be filled up with RNase-free water
to 200 µL.
2 Adjust RNA binding conditions
Add one volume of Buffer RCU to the sample (e.g.,
100 µL RCU to 100 µL sample) and mix 2 x 5 s. If
necessary, spin down gently (approx. 1 s at 1,000 x g)
to clean the lid.
3 Bind RNA
Take one NucleoSpin
placed in a Collection Tube for each preparation. Load
up to 300 µL sample mix to the column. Centrifuge for
30 s at 11,000 x g.
For volumes exceeding 300 µL, load the sample mix in
two subsequent centrifugation steps onto the column.
Place the column in a new Collection Tube (2 mL).
Maximal loading capacity of NucleoSpin
Columns is 600 µL. However, for maximum performance
loading at most 300 µL onto the column for one
centrifugation step is recommended. For larger volumes,
load the sample mix in two (or more if necessary)
successive centrifugation steps. Repeat the procedure if
larger volumes are to be processed. For high demanding
applications, the recovery rate can further be increased
as follows: Centrifuge 30 s at 2,000 x g prior to
centrifugation for 30 s at 11,000 x g.
®
NucleoSpin RNA Clean-up XS
®
RNA XS Column (light blue ring)
MACHEREY-NAGEL – 03/2021, Rev. 05
®
RNA XS
+ 1 vol. RCU
Mix
(2 x 5 s)
Load sample
mix
11,000 x g,
30 s
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