Transcription of Lipofectamine® RNAiMAX Reagent
1 Protocol Pub. No. MAN0007825 RNAiMAX ReagentProtocol OutlineA. Plate cells so they will be 60-80% confluent at the time of Prepare RNA-lipid Add RNA-lipid complexes to RNAiMAX Transfection ProtocolSee page 2 to view a typical RNAiMAX transfection Amounts96-well24-well6-wellFinal siRNA used per well1 pmol5 pmol25 pmolFinal lipofectamine RNAiMAXused per LReverse Transfection of RNAiReverse transfection is faster to perform than forward transfection and is the method of choice for high-throughput transfection. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium.
2 Because the cells and siRNA-lipid complexes are prepared on the same day, we recommended using more cells than for a regular transfection method. Scaling Up or Down Transfections Limited Product Warranty and Disclaimer DetailsPackage ContentsCatalog Number 13778-100 13778-030 13778-075 13778-150 mL15 mLStorage ConditionsStore at 4 C (do not freeze).Required Materials siRNA or miRNA (10 M stock) Opti-MEM Reduced Serum Medium Eppendorf tubesTimingPreparation: 10 minutesIncubation: 5 minutesFinal Incubation: 1-3 daysSelection GuideLipofectamine ReagentsGo online to view related Description lipofectamine RNAiMAX Transfection Reagent is a proprietary formulation for transfecting small RNAs ( , siRNA, Silencer Select siRNA, Stealth RNAi, mirVana miRNA mimics and inhibitors) into a wide range of eukaryotic Guidelines RNA- lipofectamine RNAiMAX complexes must be made in serum-free medium such as Opti-MEM Reduced Serum Medium and can be added directly to cells in culture medium, in the presence or absence of serum/antibiotic.
3 It is not necessary to remove complexes or change/add medium after transfection. Use 10 nM RNAi duplex as a starting point. BLOCK-iT Alexa Fluor Red Fluorescent Oligo (Cat. no. 14750100) can be used to determine transfection ResourcesVisit our product page for additional information and protocols. For support, visit Research Use Only. Not for use in diagnostic support, visit RNAiMAX Reagent Protocol 2013-2-Typical RNAiMAX Transfection ProcedureTransfect cells according to the following table. The transfection is designed for one RNA amount combined with one amount of lipofectamine RNAiMAX .
4 The prepared mix is enough to have triplicates (96-well), duplicates (24-well), and single well (6-well) transfections, and account for pipetting variations. For additional information on scaling your transfection reaction, see page Details1234567 Day 1 Day 2 4 Day 05 Seed cells to be 60-80% confluent at transfectionComponent96-well24-well6-wel lAdherent cells1 4 2 105 1 106 Dilute lipofectamine RNAiMAX Reagent in Opti-MEM MediumOpti-MEM Medium 25 L 50 L 150 LLipofectamine RNAiMAX Reagent L3 L9 LDilute siRNA in Opti-MEM MediumOpti-MEM Medium 25 L 50 L 150 LsiRNA (10 M) L (5 pmol)1 L (10 pmol) 3 L (30 pmol)Add diluted siRNA to diluted lipofectamine RNAiMAX Reagent (1.)
5 1 ratio)Diluted siRNA25 L 50 L150 LDiluted lipofectamine RNAiMAX Reagent25 L50 L150 LIncubateIncubate for 5 minutes at room siRNA-lipid complex to cellsComponent96-well24-well6-wellsiRNA- lipid complex per well10 L 50 L 250 LFinal siRNA used per well1 pmol5 pmol25 pmolFinal lipofectamine RNAiMAX used per LVisualize/analyze transfected cellsIncubate cells for 1 3 days at 37 C. Then, analyze transfected cells.
