Bio-rad Aurum™ Total RNA 96 Kit Manual do Utilizador descarregar pdf (Página 10)

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.

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Bio-rad Aurum™ Total RNA 96 Kit Manual do Utilizador Página 10