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The cleavage-deprotection mixture was chilled to 5°C and added to the resin, and then allowed to come to 25°C with continuous stirring over a period of 2 hrs to insure complete deprotection [25].
The mixture was chilled on ice and probe-tip sonicated (Cole Parmer) with a 1/4 in.
The overall mixture was chilled to 4 °C and TEMED (Thermo Fisher) was added to 2% v/v.
At termination of the enzymatic hydrolysis a given mixture was chilled immediately on ice and centrifuged at 5000 rpm for 10 min.
The mixture was chilled on ice and incubated (45°C, 50 min) with fivefold concentrated reverse transcriptase buffer (50 mM Tris HCl, pH 8.4, 75 mM KCl (Gibco BRL, 18064-014), 3 mM MgCl2 (Gibco BRL, 18064-014), 500 µM of each deoxynucleotide, 10 mM dithiothreitol (Gibco BRL, 18064-014) and 200 U of Superscript II, RT Moloney Murine Leukemia virus reverse-transcriptase (Gibco BRL, 18064-014).
The mixture was chilled and 1 mL of concentrated sulphuric acid added; the deep blue solution was then shaken.
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Leave it in the refrigerator until the mixture is chilled.
"I was chilled".
Refrigerate the mixture until it's chilled.
Dead Hand was chilling.
The mixture was then chilled down on ice and stored at −20°C.
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