Functional Genomics and RNAi

RNAi Tuschl Protocols
Sigma has prepared product lists for commonly used protocols from Cold Spring Harbor Protocols.

 

Selected Protocols from "RNAi, A Guide to Gene Silencing", Gregory J. Hannon.
Mammalian RNA Interference, Thomas Tuschl (Chapter 13, pg. 280-288)

NOTE: Selected protocol links below require a subscription to Cold Spring Harbor Protocols to view.		  
Selection of siRNA Sequences for Mammalian RNAi
Annealing siRNAs to Produce siRNA Duplexes
Cell Culture and Preparation of Cells in 24-Well Plates
Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes
Transfection of siRNA Duplexes
Immunofluorescence Detection of Protein Knockdown
Detection of Protein Knockdown by Western Blotting
ABSTRACT
A series of protocols is presented for targeting endogenous genes in mammalian somatic cells.


Products Available for "Annealing siRNAs to Produce siRNA Duplexes"
Protocol Material Description Product #  Product Name Add to Cart
200 mM potassium acetate P1190 Potassium acetate
   
4 mM magnesium acetate 63052 Magnesium acetate solution
   
60 mM HEPES-KOH (pH 7.4) H0887 HEPES solution
   
Ethidium bromide solution (1% w/v) aqueous E1510 Ethidium bromide solution
   
Agarose suitable for small PCR fragments A3038 Agarose
   
Sense and antisense siRNA in H2O at a conc. >80uM Custom siRNA Production of siRNA  
Gel loading buffer G2526 Gel Loading Buffer
   
5X TBE buffer T6400 Tris-Borate-EDTA buffer
   
450 mM Tris base T6066 Trizma® base
   
450 mM boric acid B6768 Boric acid
   
10 mM Na2EDTA E7889 Ethylenediaminetetraacetic acid disodium salt solution
   
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Products Available for "Cell Culture and Preparation of Cells in 24-Well Plates"
Protocol Material Description Product #  Product Name Add to Cart
Dulbecco's modified Eagle medium (DMEM) D5796 Dulbecco's Modified Eagle's Medium - high glucose
   
Fetal bovine serum (FBS) F2442 Fetal Bovine Serum
   
Penicillin and streptomycin P4458 Penicillin-Streptomycin
   
Trypsin-EDTA solution T4049 Trypsin-EDTA solution
   
Cell Culture flask (175 ml) C7046 Corning® cell culture flasks
   
Cell culture plate (24 well) CLS3337 Corning® Cell Culture Plates with CellBIND® surface
   
Coverslips S1815 SecureSlip™ glass coverslip
   
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Products Available for "Cotransfection of Luciferase Reporter Plasmids with siRNA Duplexes"
Protocol Material Description Product #  Product Name Add to Cart
Cell culture plate (24 well) CLS3337 Corning® Cell Culture Plates with CellBIND® surface
   
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Products Available for "Immunofluorescence Detection of Protein Knockdown"
Protocol Material Description Product #  Product Name Add to Cart
Hoechst 33342 B2261 bisBenzimide H 33342 trihydrochloride
   
Methanol, chilled to –10°C 494437 Methanol
   
137 mM NaCl S3014 Sodium chloride
   
7 mM Na2HPO4 S3264 Sodium phosphate dibasic
   
1.5 mM KH2PO4 60353 Potassium phosphate dibasic
   
2.7 mM KCl P9541 Potassium chloride
   
Cell culture plate (24 well) carrying knockdown cells on coverslips CLS3337 Corning® Cell Culture Plates with CellBIND® surface
   
Coverslips S1815 SecureSlip™ glass coverslip
   
Filter paper Z612820 Schleicher & Schuell® filter paper
   
Petri dish (15-cm diameter) Z231525 Duroplan Petri Dishes
   
Slides Z162701 Corning® microscope slides, frosted one side, one end
   
Tweezers (No. 7) T4912 Tweezers
   
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Products Available for "Detection of Protein Knockdown by Western Blotting"
Protocol Material Description Product #  Product Name Add to Cart
Blocking solution (3% milk powder in TBST [pH 7.4]) P4739 Phosphate buffered saline
   
Dulbecco's modified Eagle medium (DMEM) D5796 Dulbecco's Modified Eagle's Medium - high glucose
   
ECL detection kit PQ0201 ProteoQwest™ Chemiluminescent Western Blotting Kit, CPS Substrate
   
Tris-Glycine Buffer T4904 Tris-Glycine Buffer 10× Concentrate
   
0.01% SDS L4390 Sodium dodecyl sulfate
   
20% methanol 494437 Methanol
   
2X Laemmli SDS sample buffer S9788 2x SDS Sample Buffer
   
137 mM NaCl S3014 Sodium chloride
   
7 mM Na2HPO4 S3264 Sodium phosphate dibasic
   
1.5 mM KH2PO4 60353 Potassium phosphate dibasic
   
2.7 mM KCl P9541 Potassium chloride
   
Ponceau S Stain P3504 Ponceau S
   
0.2% Tween-20 P9416 TWEEN® 20
   
20 mM Tris-HCl T5941 Trizma® hydrochloride
   
150 mM NaCl S3014 Sodium Chloride
   
Trypsin-EDTA solution T4049 Trypsin-EDTA solution
   
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Product Association Disclaimer: The Sigma-Aldrich products listed for this specific protocol were selected either to match or to supplement the products listed within the actual protocol. The products/reagents from Sigma-Aldrich have been qualified for usage, but may not have been validated for this specific application. Please refer to the detailed product description on the usage of specific products of interest.