84 Gerard and D'Alessio
- If made with RNase-free labware, most solutions can be made from
reagent-grade materials and distilled water, and autoclaved. Solutions
that are heat-sensitive should be made with autoclaved, distilled water
and sterile-filtered to 0.2 ~un in disposable plasticware. - If all else fails, most aqueous buffer solutions can be treated with 0.01%
(v/v) DEPC and autoclaved. Buffers containing primary amines (such
as Tris) cannot be effectively treated by this method. - Dedicate a separate set of automatic pipets for manipulating RNA and
the buffers and enzymes used to synthesize cDNA. - Always wear disposable gloves to prevent contamination.
4.2. Materials
4.2.1. Reagents, Supplies, and Equipment - M-MLV H- RT(BRL) (SuperScript TM RT).
- E. coli DNA polymerase I.
- E. coli DNA ligase.
- E. coli RNase H.
- T4 DNA polymerase.
- pd(N) 6 (Random Hexamers) (Pharmacia LKB Biotech., Inc., Piscataway, NJ).
- pd(T)12_18 [oligo(dT)12_ls].
- [o~-32p]dCTP (>3000 Ci/mmol).
- GF/C glass fiber filters (1 x 2 cm).
- dATP, dCTP, dGTP, dTTP.
- Autoclaved 0.5- and 1.5-mL microcentrifuge tubes.
- Automatic pipets capable of dispensing 1-20 ~L and 20-200 laL.
- Autoclaved, disposable tips for automatic pipets.
- Disposable gloves.
- 37 and 16°C water baths.
- Phenol:CHCl3:isoamyl alcohol (25:24:1 [v/v/v]): Saturate a bottle of
redistilled phenol with distilled water. When the phases have separated,
remove some of the bottom layer (phenol) with a glass pipet and trans-
fer it to a clean glass bottle. Add an equal vol of CHCl3:isoamyl alcohol
(24:1 [v/v]), and store at 4°C until use. This solution should not be kept
for more than 1 wk. The water-saturated phenol should have the excess
water removed from it before it is stored at -20°C until needed again. - 10% (w/v) TCA containing 1% (w/v) sodium pyrophosphate (store at 4°C).
- Yeast tRNA, 1 mg/mL: Place 25 mg of yeast tRNA (BRL) in 2.5 mL of
0.4M NaC1-0.05M Tris-HC1 (pH 7.5); add 0.1 mL of 10% sodium
dodecyl sulfate and 1 mL of 1 mg/mL proteinase K (BRL). Incubate at
37°C for 2 h. Extract at room temperature three times with an equal vol
of phenol:CHC13:isoamyl alcohol, and precipitate the tRNA from the