96-well 24-well 6-well - Thermo Fisher Scientific

1 For support visit 3000 Reagent ProtocolProtocol OutlineA. Plate cells so they will be 70 90% confluent at the time of Prepare plasmid DNA-lipid complexes (recommend 2 doses of lipid).C. Add DNA-lipid complexes to AmountsTransfection of siRNATo transfect cells with siRNA, follow the protocol as described for DNA but do not add P3000 Reagent when diluting the siRNA (step 3).Limited Product WarrantyLife Technologies Corporation and/or its affiliate(s) warrant their products as set forth in the Life Technologies General Terms and Conditions of Sale found on Life Technologies website at If you have any questions, please contact Life Technologies at Licensing InformationThese products may be covered by one or more Limited Use Label Licenses.

2 By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses. DisclaimerLIFE TECHNOLOGIES CORPORATION AND/OR ITS AFFILIATE(S) DISCLAIM ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT, EXPRESSED OR IMPLIED, INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE, OR NON-INFRINGEMENT. TO THE EXTENT ALLOWED BY LAW, IN NO EVENT SHALL LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) BE LIABLE, WHETHER IN CONTRACT, TORT, WARRANTY, OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING BUT NOT LIMITED TO THE USE Entity: Life Technologies | Carlsbad, CA 92008 USA | Toll free in USA 2016 Thermo Fisher Scientific Inc.

3 All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified. 10 February 2016 Package ContentsCatalog NumbersL3000001L3000008L3000015L3000075L 3000150 mL5 mL 15 mLStorage ConditionsRequired Materials Store at 4 C (do not freeze). MicrOpti-MEMP lasmid DNAocentrifuge tubes ( 5 g/ L stock) Reduced Serum Medium TimingPreparation: 10 minutesIncubation: 10 15 minutesFinal Incubation: 1 3 daysSelection GuideProduct DescriptionLipofectamine ReagentsGo online to view related products. Lipofectamine 3000 Reagent is a proprietary formulation for transfecting nucleic acids into a wide range of eukaryotic cells and especially designed for hard to transfect cellsImportant Guidelines Make DNA-Lipofectamine 3000 complexes in serum-free medium such as Opti-MEM Reduced Serum Medium and add directly to cells in culture medium, in the presence or absence of serum/antibiotic.

4 It is not necessary to remove complexes or change/add medium after transfection. The amount of Lipofectamine 3000 Reagent for successful transfection varies. Start any new transfection by testing the recommended two concentrations of Lipofectamine 3000 Reagent to determine an optimum ResourcesVisit our product page for additional information and protocols. For support visit Research Use Only. Not for use in diagnostic 3000 ReagentUSER GUIDED ocument Part No. 100022234 Publication No. MAN0009872 Rev per wellP3000 Reagent per wellLipofectamine 3000 Reagent per well96-well100 and L24-well500 ng1 and L6-well2500 ng5 and LLipofectamine 3000 Reagent ProtocolFor support visit cells according to the following table.

5 Use the indicated volume of DNA and P3000 Reagent with each of the two volumes of Lipofectamine 3000 (when performing optimization). Each reaction mix volume is for one well and accounts for pipetting variations. Scale volumes proportionally for additional Details (Two Reaction Optimization)Day 01 Seed cells to be 70 90% confluent at transfectionComponent96-well24-well6-wel lAdherent cells1 4 2 1 106 Day 12 Diluted Lipofectamine 3000 Vortex 2 3 secDilute Lipofectamine 3000 Reagent in Opti-MEM Medium (2 tubes) Mix wellOpti-MEM Medium5 L 225 L 2125 L 2 Lipofectamine 3000 and and and L3 Diluted DNAP repare master mix of DNA by diluting DNA in Opti-MEM Medium, then add P3000 Reagent Mix wellOpti-MEM Medium10 L50 L250 LDNA ( 5 g/ L) g1 g5 gP3000 Reagent (2 L/ g DNA) L2 L10 L4 Add Diluted DNA to each tube of Diluted Lipofectamine 3000 Reagent (1.)

6 1 ratio)Diluted DNA (with P3000 Reagent)5 L25 L125 LDiluted Lipofectamine 3000 Reagent5 L25 L125 L510-15 IncubateIncubate for 10 15 minutes at room DNA-lipid complex to cellsComponent (per well)96-well24-well6-wellDNA-lipid complex10 L50 L250 LDNA amount100 ng500 ng2500 ngP3000 L1 L5 LLipofectamine 3000 Reagent and and and LDay 2 47 Visualize/analyze transfected cellsIncubate cells for 2 4 days at 37 C. Then, analyze transfected cells.