Protein Expression

Gene Silencing

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View the entire protocol on BioSupplyNet.com.

View the entire protocol "Production of Endoribonuclease-Prepared Short Interfering RNAs (esiRNAs) for Specific and Effective Gene Silencing in Mammalian Cells" at BioSupplynet.com

Anne-Kristin Heninger and Frank Buchholz

ABSTRACT
The mechanism of RNA interference has emerged as a practical tool to study loss of function in many organisms. To make the method of gene knockdown suitable for mammalian cells, short interfering double-stranded RNAs (siRNAs) need to be applied. This protocol describes a straightforward, low-cost method, which uses recombinant Escherichia coli RNase III to digest long dsRNA into endoribonuclease-prepared short interfering RNAs (esiRNAs). Advantages of this technology are the high efficiency and specificity of the resulting esiRNA and its usefulness for not only small-scale applications, but also high-throughput loss-of-function analyses. Another great asset of esiRNA is its flexibility in design, using Web-based tools such as DEQOR, or predesigned esiRNA sequences from the database RiDDLE. PCR products flanked with T7 promoter sequences are generated, transcribed, and annealed. The resulting long dsRNA is enzymatically digested into a pool of overlapping esiRNAs, which are subsequently spin-column-purified.

Products Available for this Protocol
Protocol Material Description Product # Product Name Add to Cart
Agarose A4718 Agarose, High resolution, DNase, RNase, NICKase, none detected
dNTP mix D7295 Deoxynucleotide Mix, 10 mM, Molecular Biology Reagent
Tris-Cl T5941 Trizma® hydrochloride, for molecular biology, Biotechnology Performance Certified, cell culture tested, ≥99.0% (titration)
EDTA E5134 Ethylenediaminetetraacetic acid disodium salt dihydrate, for molecular biology, ≥99%
MgCl2 M4880 Magnesium chloride, insect cell culture tested
DTT D9779 DL-Dithiothreitol, for molecular biology, ≥98% (TLC), ≥99% (titration)
NaCl S6191 Sodium chloride, Biotechnology Performance Certified, ≥99.5% (titration), Cell Culture Tested
KCl P9541 Potassium chloride, for molecular biology, ≥99%
Glycerol G5516 Glycerol, for molecular biology, ≥99%
H2O W4502 Water, Molecular Biology Reagent
Ethanol E7148 Ethanol, for molecular biology, 190 proof
Isopropanol I9782 Isopropyl alcohol, meets EP, BP, USP testing specifications
Ficoll F2637 Ficoll®, for molecular biology, lyophilized powder
Orange G (acid orange 10) O7252 Orange G, certified by the Biological Stain Commission
DNA marker with fine resolution (e.g., 25-bp DNA ladder) P1598 PCR 20 bp Low Ladder, buffered aqueous solution
Red DNA polymerase D2812 REDTaq® Genomic DNA Polymerase, without MgCl2
Q-Sepharose FastFlow Q1126 Q Sepharose® Fast Flow, preswollen, 45-165 µm (wet), pore size ~4,000,000 Da exclusion limit

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