Bio-Rad Aurum™ Total RNA 96 Kit - Manual (Page 10)

6
Disruption and Homogenization
Proper disruption and homogenization of the starting materials are required to
ensure complete lysis of the cells and to reduce the viscosity of the cell lysates.
This procedure uses repeated pipetting up and down through micropipettor tips
to aid in lysing the cells, as well as to prepare homogeneous lysates prior to
loading onto the RNA binding plate. Make sure that cell lysates are thoroughly
homogenized (i.e. the viscosity has been considerably reduced); failure to do so
may result in well clogging and reduced RNA purity.
Section 6
Vacuum Manifold Setup and Use With
96-Well Plates
Guidelines for Vacuum Format
• The recommended operating range is –17 to –23 inHg (see Table 2 for
pressure unit conversions). Do not exceed –25 inHg when performing this
protocol. A vacuum regulator is strongly recommended to establish the
appropriate negative pressure.
Table 2. Pressure unit conversions.
To convert from inches of mercury (inHg) to: Multiply by:
millimeters of mercury or torr (mmHg, torr) 25.4
millibar (mbar) 33.85
atmospheres (atm) 0.03342
pounds per square inch (psi) 0.4912
kilopascals (kPa) 3.385
• The gasket used on the manifold allows the RNA binding plate to self-seat
whenever vacuum is applied, without pressing down on the plate. However,
under certain conditions, gentle downward force may be required to ensure
that the plate forms a seal with the manifold.
• When applying a vacuum to the manifold, increase the negative pressure
gradually by slowly closing the vacuum regulator over a 5–10 sec period. This
will promote uniform movement of solutions through the wells of the binding
plate, minimizing sample spraying and cross-contamination during elution.
• During purge steps, the negative pressure within the vacuum manifold may
drop to below –15 inHg. This is normal and does not require corrective action.