( nt pellet to a fresh 50 ml...)Simple and Rapid Isolation of Plant RNA,,, Using the Eppendorf Perfect RNA, Eukaryotic, Maxi Kit

nt pellet to a fresh 50 ml tube without any carryover of the cell debris. If the pellet is loose, the supernatant should be filtered through an RNase-free Miracloth (Callbiochem).

Add 18 ml 70% ethanol to lysate and completely mix via very gentle, repeated inversion.

Add 4 ml well-mixed Perfect RNA Binding Matrix Solution and mix via gentle inversion.

Place two Perfect RNA Maxi Spin Columns into 50 ml centrifuge tubes and transfer lysate/Binding Matrix mixture to two Spin Column assemblies.
Note: A modified preparation procedure for difficult tissues is described in the detailed protocol Step 6.

Centrifuge for 5 to 10 min. at 2,000 x g. Discard filtrate.

Add 10 ml Wash Solution I to each Spin Column and centrifuge for 5 min. at 2,000 x g. Carefully transfer Spin Columns to fresh centrifuge tubes without splashing Wash Solution I.

Add 10 ml Diluted Wash Solution II (ethanol not provided) to each Spin Column and centrifuge 5 min. at 2,000 x g. Carefully transfer dry Spin Columns to RNase-free 50 ml Collection Tubes (provided).

Add 1 ml Molecular Biology Grade Water to each Spin Column and vortex assemblies briefly.

Incubate at 50C for 5 min.

After incubation, vortex for 5 sec. and immediately centrifuge for 5 min. at 2,000 x g. Store recovered total RNA at 80C.

Results

About 200 g RNA of each were obtained from 1 g source material of Arabidopsis leaves, flowers, or pods, while 5 g yielded about 800 g RNA, and 10 g yielded 850 g RNA. With respect to maize, the yields were somewhat lower: 180 g, 560 g, a
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