peqGOLD HP Total RNA Kit - PEQLAB Biotechnologie GmbH

3. 3. Load Load and and bind
bind
Transfer the upper aqueous phase into a new 1.5 ml centrifuge tube. Add an equal
volume 70 % Ethanol to the lysate and mix thoroughly by vortexing. Place a PerfectBind
RNA Column in a 2 ml Collection Tube and add 750 µl of the lysate directly to the
membrane. Centrifuge the PerfectBind RNA Column/Collection Tube assembly at
10.000 x g for 15 sec. Pour off the flow-through liquid. Repeat this step by loading the
remaining lysate to the column.
A precipitate may form on addition of 70 % ethanol. This will not interfere with RNA purification.
Vortex and add the entire mixture to the column. The maximum capacity of the spin column is
750 µl, larger volumes can be loaded successively by repeating the loading procedure. However,
the total binding capacity of a spin column is 100 µg RNA.
4. 4. 4. Wash Wash Wash II
I I
Add 500 µl RNA Wash Buffer I to the PerfectBind RNA Column and centrifuge for 15 sec
at 10.000 x g. Place the PerfectBind RNA Column in a fresh 2 ml Collection Tube.
Discard the flow-throw liquid and the used Collection Tube.
5. DNase DNase I I Digestion Digestion (optional)
Since PerfectBind RNA resin and spin-column technology actually removes most of DNA
without the DNase treatment, it is not necessary to do DNase digestion for most
downstream applications. However, certain sensitive RNA applications might require
further DNA removal. Following steps provide on-membrane DNase I digestion (Order
No. 12-1091).
a. For each PerfectBind RNA Column, prepare this DNase I digestion reaction mix:
DNase I Digestion Buffer 73.5 µl
RNase-free DNase I (20 Kunitz units/µl) 1.5 µl
Total volume 75 µl
Note:
1. DNase I is very sensitive for physical denaturation, so do not vortex this DNase I mixture!
Mix gently by inverting the tube. Prepare the fresh DNase I digestion mixture directly before
RNA isolation.
2. DNase I Digestion Buffer is supplied with RNase-free DNase set. Standard DNase buffers
are not compatible with on-membrane DNase digestion!
b. Pipet 75 µl of the DNase I digestion reaction mix directly onto the surface of
PerfectBind RNA resin in each column. Make sure to pipet the DNase I digestion
mixture directly onto the membrane. DNase I digestion will not be complete if some of
the mix stick to the wall or the O-ring of the PerfectBind RNA Column.
c. Incubate at room temperature (25 - 30 °C) for 15 minutes.
peqGOLD HP Total RNA Kit Ord.-No.: 12-6935/12-6936
V.0111 PEQLAB Biotechnologie GmbH 14