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Primer Extension Analysis Prepare RNA from 2-5 OD600 units of yeast collected at mid-log (OD600 = 0.1-0.25). Do not
 

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 g/ml). For 25S rRNA, 1 g = ~1 pmol.
Dilute oligonucleotide primer to a concentration of ~10 pmoles/l.
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 g of RNA (instead of 1 g) for standard primer extension.
Digest 2 g RNA in 20 l 50 mM Na2CO3, pH 9.0. 100 mM Na2CO3 = 1.06 g in 100 mls (check
pH). Heat to 90C for 3-5 minutes (try different times). Add KOAc, pH 5, and EtOH precipitate.
1. Set up reaction: Standard Primer Extension CDP Assay
1 l RNA (1 g, ~1 pmol) 2 l RNA (2 g, ~2 pmol)
1 l primer (10 pmol) 2 l primer (20 pmol)
7 l ddH2O 14 l ddH2O
1 l "5X" Promega Buffer 2 l "5X" Promega Buffer
10 l Final Volume 20 l 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).

  

Source: Aris, John P. - Department of Anatomy and Cell Biology, University of Florida

 

Collections: Biology and Medicine