Please cite as: CSH Protocols; 2006; doi:10.1101/pdb.prot4325

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protocolProtocol

Preparation of siRNAs from Drosophila Embryo Extracts

Luyun Huang, John Gledhill, and Craig E. Cameron

This protocol was adapted from "RNA-Dependent RNA Polymerase in Gene Silencing," contributed by Luyun Huang, John Gledhill, and Craig E. Cameron, in RNAi: A Guide to Gene Silencing (ed. Hannon). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2003.

The first 15% of the full text of this article appears below.


INTRODUCTION

siRNAs produced upon the addition of dsRNA to Drosophila embryo extract are enriched in a micrococcus-nuclease-resistant fraction. After proteinase K treatment and dephosphorylation with calf intestinal phosphatase, these siRNAs mediate efficient RNAi in vitro.


MATERIALS

Reagents

caution{alpha}-Amanitin

ATP

caution CaCl2

Calf intestinal alkaline phosphatase

Creatine kinase

Creatine phosphate

caution Dithiothreitol (DTT) (5 mM)

Drosophila embryo S100 extract . . . [Full Text of this Article]

Equipment


METHOD


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