UM_TotalRNA_NucleoZOL(11)

user's manuals about RNA or or

5

Wash RNAAdd 400–600 μL 75 % ethanol when working with 1.5 mL tubes. For larger tubes, add 500 μL 75 % ethanol per 1 mL supernatant used for precipitation.Centrifuge for 1–3 min at 4,000–8,000 x g. Remove ethanol using a micropipette. Repeat washing step. Do not dry the pellet.Add 400–600 μL 70 % isopropanol when working with 1.5 mL tubes. For larger tubes, add 500 μL 70 % isopropanol per 1 mL supernatant used for precipitation.

Centrifuge for 1–3 min at 4,000–8,000 x g. Remove ethanol using a micropipette. Repeat washing step. Do not dry the pellet.

Drying the RNA pellet may lead to a decrease in solubility.6

Reconstitute RNA

Dissolve the RNA pellet in RNase-free water to obtain an RNA concentration of 1–2 μg/μL for the large RNA fraction and about 0.1 μg/μL for the small RNA fraction. Vortex the sample for 2–5 min at room temperature for efficient solubilization.

For accurate determination of RNA concentration by OD measurement with a cuvette, dilute RNA in RNase-free water with a slightly alkaline pH, 1 mM NaOH or buffer with a pH > 8 (e.g., Elution Buffer AE, see ordering information 6.3). Distilled water typically has a pH < 7.