Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4154

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Yeast RNA Isolation: Large-Scale

David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern

This protocol was adapted from "Yeast RNA Isolations," Techniques and Protocols 6, in Methods in Yeast Genetics, 2005 edition, by David C. Amberg, Daniel J. Burke, and Jeffrey N. Strathern. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2005.

The first 100 words of the full text of this article appear below.


INTRODUCTION

This protocol describes large-scale yeast RNA isolation. It is designed to yield more than 10 mg of total nucleic acid and 300-400 µg of poly(A)-selected mRNA from each 500-ml culture.


MATERIALS

Reagents

Fresh overnight culture of an appropriate yeast strain

caution Chloroform (optional, see Step 9)

caution Cycloheximide (optional, see Step 2)

Ethanol, 80% and 100%

HEPES (10 mM, pH 7.0)

recipe LETS buffer

recipe LiCl, 5 M

Oligo(dT) column

recipe Oligo(dT) column loading buffer

caution Phenol, equilibrated with LETS buffer

caution Phenol:chloroform:isoamyl alcohol(25:24:1)

RNase inhibitor (optional, see Step 4)

recipe SDS, 10%

recipe caution Sodium acetate (3 M)

recipe YPD or SC medium

Equipment

Centrifuge bottles, large (Sorvall GS3 or equivalent)

Centrifuge tubes . . . [Full Text of this Article]


METHOD


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