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Summary: 60
Primer Extension Analysis
· Prepare RNA from 2-5 OD600 units of yeast collected at mid-log (OD600 = 0.1-0.25). Do not
resuspend RNA pellet in proteinase K + SDS. LiCl present in RNA samples must be removed,
otherwise it will inhibit reverse transcriptase. Determine RNA concentration by measuring the
A260 of a diluted RNA sample (A260 of 1 = 40 µg/ml). For 25S rRNA, 1 µg = ~1 pmol.
· Dilute oligonucleotide primer to a concentration of ~10 pmoles/µl.
· Prepare in vitro transcribed RNA control for concentration dependent pause (CDP) assay.
· For CDP assay, set up tubes "4", "20", and "100" in advance (see below).
· Partial alkaline hydrolysis. Use 2 µg of RNA (instead of 1 µg) for standard primer extension.
Digest 2 µg RNA in 20 µl 50 mM Na2CO3, pH 9.0. 100 mM Na2CO3 = 1.06 g in 100 mls (check
pH). Heat to 90°C for 3-5 minutes (try different times). Add KOAc, pH 5, and EtOH precipitate.
1. Set up reaction: Standard Primer Extension CDP Assay
1 µl RNA (1 µg, ~1 pmol) 2 µl RNA (2 µg, ~2 pmol)
1 µl primer (10 pmol) 2 µl primer (20 pmol)
7 µl ddH2O 14 µl ddH2O
1 µl "5X" Promega Buffer 2 µl "5X" Promega Buffer
10 µl Final Volume 20 µl Final Volume
Set up reactions in 0.5 ml tubes. Use Promega buffer at "0.5X" (25 mM Tris-HCl, pH 8.3, 25
mM KCl, 5 mM MgCl2, 5 mM DTT, 0.25 mM spermidine).
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