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RNeasy Mini Handbook 10/2019
47
Buffer RLT and Buffer RW1 contain a guanidine salt and are therefore not compatible
with disinfecting reagents containing bleach. See page 7 for safety information.
Perform all steps of the procedure at room temperature. During the procedure, work
quickly.
Perform all centrifugation steps at 20–25°C in a standard microcentrifuge. Ensure that
the centrifuge does not cool below 20°C.
Things to do before starting
β-Mercaptoethanol (β-ME) must be added to Buffer RLT before use. Add 10 µl β-ME per
1 ml Buffer RLT. Dispense in a fume hood and wear appropriate protective clothing.
Buffer RLT containing β-ME can be stored at room temperature for up to 1 month.
Alternatively, add 20 µl of 2 M dithiothreitol (DTT) per 1 ml Buffer RLT. The stock solution
of 2 M DTT in water should be prepared fresh or frozen in single-use aliquots. Buffer RLT
containing DTT can be stored at room temperature for up to 1 month.
Buffer RPE is supplied as a concentrate. Before using for the first time, add 4 volumes of
ethanol (96–100%) as indicated on the bottle to obtain a working solution.
If performing optional on-column DNase digestion, prepare DNase I stock solution as
described in Appendix D (page 82).
Procedure
1. Excise the tissue sample from the animal or remove it from storage. Remove RNAprotect
stabilized tissues from the reagent using forceps. Determine the amount of tissue. Do not
use more than 30 mg.
Weighing tissue is the most accurate way to determine the amount.
Note: If the tissues were stored in RNAprotect Reagent at −30 to −15°C, be sure to
remove any crystals that may have formed.
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