Bio-Rad PureZOL™ RNA Isolation Reagent User Manual
Page 9
Section 5
Protocol
Carry out all steps at room temperature unless otherwise indicated.
RNase-free disposable polypropylene tubes should be used
throughout the procedure. The entire procedure should take less
than 1 hour.
1. Disrupt and homogenize the sample using the following
suggestions depending on the sample type:
Cells Grown in a Monolayer
Cells grown in a monolayer should be lysed with PureZOL
directly in the culture dish. Aspirate the culture medium and
immediately add 1 ml of PureZOL to a 10 cm
2
dish. Pipet up
and down several times. The amount of PureZOL added is
dependent on the area of culture dish (1 ml per 10 cm
2
) and
not on cell number. Insufficient volumes of PureZOL may result
in DNA contamination. Note: Do not wash cells prior to the
addition of PureZOL as this could increase the possibility of
mRNA degradation.
Suspension Cells (Mammalian, Plant, Bacterial, or Yeast)
Pellet the cells by centrifuging at 3,000–5,000 x g for 2 minutes.
Immediately lyse by adding 1 ml of PureZOL to 1 x 10
7
cultured
mammalian and plant cells, 2.4 x 10
9
of Gram-positive or
Gram-negative bacteria, or 3.0 x 10
7
of yeast in a suitable
sized tube. Pass the lysate through a pipet or an 18-gauge
needle and syringe several times. To improve the efficiency of
the cell lysis process, a rotor-stator or bead mill homogenizer is
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