Use this procedure to reverse transfect stealth rnai or sirna into mammalian cells in a 24well. Plate cells so they will be 6080% confluent at the time. If you want to use lipofectamine rnaimax for your cotransfections, perform a reverse transfection with the following modifications. Reverse transfection magnetofectamine protocol v 1. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Reverse transfection of rnai reverse transfection is faster to perform than forward transfection and is the method of choice for highthroughput transfection. For reverse transfection, cells in optimal growth phase are prepared so that it is possible to. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations.
A typical forward transfection protocol using transit2020 transfection reagent can be found here pdf. Reverse transfection an overview sciencedirect topics. Magnetofectamine is the association of the famous lipofectamine 2000 transfection reagent from invitrogen, with the unique magnetofection based. Reverse transfection combines rna, transfection reagent, and cells in an altered sequence compared to traditional lipofectamine 2000 transfection protocols. The amount of lipofectamine 2000 reagent required for successful transfection varies depending on the cell type and passage number. Has anyone here attempted reverse transfection using lipofectamine 3000 reagent. Rnaimax reverse transfections lipofectamine thermo. Version 1, 41 8 during the incubation of the printed slides with the transfection reagent, harvest hek293t cells and suspend 10 x 106 cells in 25 ml medium dmem with 10% ifs, 50 unitsml penicillin and 50 gml streptomycin. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. Reverse transfection of stealth rnai or sirna using lipofectamine.
Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. Transfecting sirna using lipofectamine rnaimax pin. Forward and reverse transfection protocols mirus bio. Transfection protocols thermo fisher scientific in. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Perform reverse transfection by preparing complexes inside the wells, and then adding cells and medium. In short, a different rnai molecule is put in each well prior to transfection and combined with diluted lipofectamine 2000 to form complexes in each well. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.
For cotransfections of plasmid dna and stealth rnai or sirna into mammalian cells, we recommend using lipofectamine 2000 catalog no. Has anyone here attempted reverse transfection using. Reverse transfection of stealth rnai or sirna using. This protocol is based on that from life technologies called. Lipofectamine 2000 reagent thermo fisher scientific. Transfection and genome engineering fisher scientific. We have found that effectene, lipofectamine 2000 and fugene 6 work. Transfection reagents product protocols thermo fisher.
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