You can generally use other serumfree medium instead of optimem. There are several ways in which to introduce cas9guide rna complexes into cells. Invitrogen lipofectamine rnaimax transfection reagent 0. Make dnalipofectamine 3000 complexes in serumfree medium such as optimem reduced serum medium and add directly to cells in culture. Lipofectamine contains lipid subunits that can form. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Transfection protocol lipofectamine 3000 transfection reagent t84. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells and provides the following advantages. Rnaimax reverse transfections lipofectamine thermo fisher.
Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Transfection of cas9 rnp ribonucleoprotein into adherent. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. I use 24well plates for transfections in triplicates. Plate cells so they will be 7090% confluent at the time of transfection. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects.
Lipofectamine rnaimax reagent protocol 20 typical rnaimax transfection procedure transfect cells according to the following table. Lipofectamine rnaimax is a proprietary rnaispecific cationic lipid formulation designed specifically for the delivery of sirna and mirna into all cell types. 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 cells important guidelines. The cell density per well and amount of lipofectamine rnaimax used per well were as follows. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. L 5 5 incubate incubate for 5 minutes at room temperature. Aug 07, 2012 in this video, we will perform an sirna transfection experiment using lipofectamine rnaimax reagent. A sample protocol is listed here for transfection experiments performed in 6well plates. As a result, sirna can be delivered into hes cells more efficiently by lipofectamine rnaimax compared with the other two transfection reagents and high efficient knockdown of target genes was. Lipofectamine plasmid transfection bridges lab protocols. Combine the diluted rnai duplex with the diluted lipofectamine rnaimax. The crisprmax transfection reagent has low cell toxicity, and provides the cleavage efficiency of electroporation with ease of scalability. Reverse transfection protocol acea biosciences inc. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection.
See page 2 to view guidelines for transfection of sirnas using lipofectamine. L final component per well 384well 96well 24well sirna 30 nm final 1. Lipofectamine 3000 protocol written out with timing. Dilute lipofectamine3000 reagent in optimem medium 2 tubes, mix well follow the instructions as indicated. Transfection protocol on the day of transfection, which should be 1 day following cell plating, perform the following steps, which have been optimized for a single well of a 24well plate using invitrogen lipofectamine 3000 transfection reagent. Lipofectaminea rnaimax reagent thermo fisher scientific. Can anybody give me exact protocol for sirna transfection using. Mix lipofectamine rnaimax gently before use, then dilute 1 l in 50 l optimem i reduced serum medium. Zhao m1, yang h, jiang x, zhou w, zhu b, zeng y, yao k, ren c.
Lipofectamine 2000 and lipofectamine rnaimax transfection reagents introduction life technologies is committed to providing researchers and scientists the best products, enabling cutting edge technology while keeping the environment in mind. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. In this video, we will perform an sirna transfection. Lipofectamine rnaimax reagent thermo fisher scientific us. For additional information on scaling your transfection reaction, see page 4. Lipofectamine rnaimax transfection reagent gives maximal knockdown and excellent cell viability across a 10fold concentration range of the reagent figure 2. Cells were transfected with lipofectamine rnaimax complexed with silencer select sirna at 30 nmwell. The transfection is designed for one rna amount combined with one amount of lipofectamine rnaimax. Genscript recommends using lipofectamine 2000 for all transfections. This protocol is optimized for transfection in a 6wellin. Rna interference methodology suppresses specific gene expression, thus mimicking lossoffunction mutation and enabling in vitro and in vivo gene function analysis.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. How much lipofectamine rnaimax shoiuld be used for sirna. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. However, delivery of the cas9 rnps by neon electroporation was effective across all different cell lines tested and often was the only method that could produce indels. Lipofectamine 3000 protocol written out with timing specifics. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Prewarm 50ml of optimum stored in cold room at 4c 10min. Reverse transfection is possible but it is recommended to use the 2. Gfp plasmid to sirna while doing lipofectamine 2000 transfection. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Add 6l of lipofectamine2000 from biostores and 100 l of. Invitrogen lipofectamine 3000 transfection reagent 1. Lipofectamine 3000 reagent is suitable for novel genome editing applications. Follow the transfection reagent protocol for seeding cells.
Transfection protocol lipofectamine 3000 transfection reagent t84 lung cancer cells complete growth medium component cat. Feb 18, 2016 lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of cell lines. Use this procedure to reverse transfect stealth rnai or sirna into mammalian cells in a 24well format for other formats, see. See page 2 to view a typical rnaimax transfection procedure. Enhanced and simple transfection protocol for a variety of cell lines. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Transfection protocol lipofectamine 3000 transfection reagent. Lipofectamine crisprmax transfection reagent is a proprietary formulation for transfecting cas9 nucleasegrna complex into a wide range of eukaryotic cells. Highest transfection efficiency in many cell types and formats e. This makes lipofectamine rnaimax reagent easy to optimize for the lowest sirna concentration while reducing cytotoxicity in your experimental system. As nehad pointed out, you can do reverse transfection with lipofectamine 2000 or rnaimax. The instructions are recommending that you do this in duplicate with the two indicated different concentrations of lipofectamine 3000.
Can anyone recommend a protocol for transfection of hepg2. For your convenience, the essential components of this protocol are now available in the. For your convenience, the essential components of this protocol are now available in the gibco prostate cancer starter kit. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Refer to the cell lines database for a list of cell types successfully transfected. Can anybody give me exact protocol for sirna transfection using rnaimax.
Sep 25, 2007 following on from other products in the lipofectamine range, lipofectamine ltx is a lipid based reagent for transfecting dna and vector based rnai into eukaryotic cells. Lipofectamine crisprmax is more robust than lipofectamine rnaimax and several other transfection reagents in delivery of cas9 proteingrna complexes into a variety of cell lines. The protocol indicated below is a reverse transfection protocol. With its ability to transfect a broad range of cell types, lipofectamine rnaimax reagent, combined with the invitrogen blockit alexa fluor red fluorescent control, gives you the most versatile approach to all of your knockdown experiments table 1, figure 3. Protocol 6 well dish plate cells the day before so that they are at 9095% confluence in 2 ml media without antibiotics. For many adherent cell types, we have observed that lipofectamine rnai max from thermo gives close to optimal results and recommend it for starting. Invitrogen lipofectamine rnaimax transfection reagent. It is used to increase the transfection efficiency of rna.
Can anyone recommend a protocol for transfection of hepg2 cells with sirna and lipofectamine. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Lipofectamine rnaimax transfection reagent provides the highest transfection efficiencies on the widest variety of cell types for sirnamediated gene knockdown experiments. Transfect cells according to the manufacturers protocol. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.
To transfect cells with sirna, follow the protocol as. This protocol is optimized for transfection in a 6well in vitro culture plate format to perform transfection in different cell culture formats, refer to table2 and invitrogen lipofectamine rnaimax protocol. Transfecting sirna using lipofectamine rnaimax pin. The transfection is designed for one rna amount combined. Scaling up or down lipofectamine 3000 reagent transfections. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. Transfection protocol transfection procedure this procedure is designed for sirna in combination with lipofectamine rnaimax. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate.
Lipofectamine rnaimax reagent protocol thermo fisher scientific. Lipofectamine ltx is an improvement on other invitrogen transfection reagents as the protocol has been simplified into a simple single tube method that can be scaled up. Lipofectamine rnaimax offers a simple protocol no need to remove transfection complexes, or change or add medium following transfection. Mix gently and incubate for 1020 minutes at room temperature. From our experiences, we noticed that lipofectamine rnaimax is superior to lipofectamine 2000 to the extent of cytotoxicity and rnai efficiency, and that cytotoxicity of lipofectamine 2000 varies lot by lot. Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Lipofectamine ltx transfection reagent from invitrogen. Lipofectamine rnaimax rnaimax, a new cationic lipofection reagent, has shown higher efficiency in delivering mirna into many stem cells and cell lines zhao et al. This protocol is optimized for transfection in a 6well in vitro culture plate format to perform. How to perform sirna transfection with lipofectamine rnaimax protocol. I am thinking about the cell density or the lipofectamine rnaimax amount that i should use. Lipofectamine 3000 transfection reagent c4 prostate. Cas9 nuclease may be used in vivo to create targeted genome modifications.
Lipofectamine rnaimax reagent easily outperforms other sirna transfection reagents. Reverse transfection an overview sciencedirect topics. Plate cells so they will be 6080% confluent at the time of transfection. Lipofectamine rnaimax is used only for small rnas, such as sirnas. Though this protocol has been optimized using the a549 human nonsmall cell lung cancer cell line, assay conditions may require additional optimization if different cell lines are. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. This protocol is for a basic assay using an xcelligence rtca instrument to monitor the effects of sirna knockdown. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. To perform transfection experiments in other cell culture plates, simply multiply the.
High knockdown levels of target genes can be achieved with as little as 1 nm sirna. Prepare plasmid dnalipid complexes recommend 2 doses of. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Lipofectamine 2000 and lipofectamine rnaimax transfection. Invitrogen have added a new product, lipofectamine ltx, to their extensive range of transfection products. For your convenience, the essential components of this protocol are now available in the gibco lung cancer starter kit. Transfection protocol lipofectamine 3000 transfection. Improved delivery of cas9 proteingrna complexes using. It increases the likelihood of successful cleavage and recombination with talens or crisprs through highly efficient transfection, ultimately maximizing the efficiency of genetic modifications and simplifying the downstream processes. Lipofectamine 2000 transfection reagent from invitrogen. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Invitrogen lipofectamine 3000 transfection reagent l3000015 print share invitrogen lipofectamine 3000 transfection reagent.
752 214 1003 540 1219 638 869 1149 586 1291 1302 545 1517 923 1105 1229 924 899 1044 1566 542 1566 1184 5 506 1150 1281 408 1382 1425 843 1043 158 694 572 741