Once you merge pdfs, you can send them directly to your email or download the file to our computer and view. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format. Invitrogen lipofectamine 2000 transfection reagent life. Rnaimax transfection reagent 778030, invitrogen, waltham, ma, usa, lipofectamine 3000. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and. Life technologies corporation andor its affiliates warrant their products as set. 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.
Mix optimem and lipofectamine in another eppendorf 32 l lipofectamine in 0,8 ml optimem 3. Dilute 4 amounts of lipofectamine 2000 reagent in optimem medium. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas \e. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with.
Transfection using effectene, lipofectamine, and polyfect reagents many reagents are now available for optimization of cell transfection efficiency. Invitrogen 11668019 view more versions of this product. Change media to fresh dmem, 10% fcs no antibiotics plate change media per well ml 6well plate 2 100 mm plates 10 150 mm plates 22. Lipofectamine 3000 transfection reagent thermo fisher scientific. L in optimem invitrogen and incubated for 15 min at room temperature. Once files have been uploaded to our system, change the order of your pdf documents. Lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna. Mix dna, plus reagent invitrogen and optimem combination well.
I use 24well plates for transfections in triplicates. Despite this, a satisfactory mechanismbased explanation of their. Lipofectamine 2000 transfection reagent from invitrogen. Use the recommended lipofectamine 2000 cd reagent amount. The highest transfection efficiency in many cell types and formats e. Cas9 nuclease may be used in vivo to create targeted genome modifications. Advanced transfection with lipofectamine 2000 reagent. Rearrange individual pages or entire files in the desired order. Lipofectamine 2000 transfection reagent thermo fisher scientific.
Versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines. Systematic screening of commonly used commercial transfection. 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 transfection reagent is one of our firstgeneration, cationiclipid transfection reagents formulated for the transfection of dna into eukaryotic cells. How can i optimize the transfection efficiency using. Fibroblast 3t3, cos7 myoblast c2c12, l6 crl1458 hepatocyte hepg2, huh7 erythroleukemic cell k562. According to our results, lipofectamine 3000, fugene and rnaimax showed high. 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. The vector contained an ofp reporter gene and was transfected with lipofectamine 2000 or lipofectamine 3000 reagent into a u2os and b hepg2 cell lines. Rna was mixed with lipofectamine rnaimax invitrogen in a ratio of 1.
Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Plate cells so they will be 7090% confluent at the time of transfection. Photograph of expected results dna lipofectamine scaling up or down. Mix optimem with dna in an eppendorf 510 g dna0,8 ml optimem 2. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine transfection reagent thermo fisher scientific. Lipofectamine 2000 is then added to culture medium within a sterile tube along with the dna at a 3. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Invitrogen lipofectamine 3000 transfection reagent from. Combine diluted dna and lipofectamine, mix and incubate at room temperature for 20 min. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment.
There are several ways in which to introduce cas9guide rna complexes into cells. Transfecting sirna using lipofectamine raiax pin liu, 2017 declaration. Refer to the cell lines database for a list of cell types successfully transfected. Since launching in 1993, invitrogen lipofectamine reagents have become the most. Researchers use lipofectamine 2000 reagent for sirna and shrnabased. Lipofectamine 3000 lipofectamine 2000 competitor fhd lipofectamine 2000 lipofectamine 3000 competitor 1 competitor 2 gststat. The following cell lines have been successfully transfected with the indicated transfection reagents.
Different cell lines were transfected with a gfp reporter vector using the indicated transfection reagents. Add diluted dna to each tube of diluted lipofectamine 2000 reagent 1. Refer to the transfection collection and the invitrogen transfection guide available at for a list of cell lines and cell types that have been successfully transfected. For additional information on scaling your transfection reaction, see page 4.
Mar 27, 2007 lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Make sure that cells are greater than 90% confluent and vary dna g. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure. For the cell lines easy to transfection, you can use either of the procedures reverse or forward. Dilute your dna and lipofectamine mix in serum free medium. Protocol 6 well dish plate cells the day before so that they are at 9095% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. For other formats, see scale up or down transfections. Combine diluted dna with diluted lipofectamine 2000 by adding the dna dilution to the lipofectamine solution.
Lipofectamine 2000 reagent thermo fisher scientific. Invitrogen lipofectamine 2000 transfection reagent. Invitrogen lipofectamine 2000 transfection reagent 1. The protocal is from thermo fisher scientific important guidelines for transfection 1. Lipofectamine rnaimax is proprietary animal originfree, chemically defined rnaispecific cationic lipid formulation designed specifically for delivery of sirna and mirna into all cell types. Lipofectamine 2000 cd reagent thermo fisher scientific. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Invitrogen lipofectamine rnaimax transfection reagent. Protocol for dna transfection and luciferase assay using.
Lipofectamine plasmid transfection bridges lab protocols. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Transfection of cas9 rnp ribonucleoprotein into adherent. Highest transfection efficiency in many cell types and formats e. Lipofectamine 3000 reagent is suitable for novel genome editing applications.
However, after 48 hours of transfections majority of the cells. Mix the lipofectamine 2000 gently and dilute 10 l in 0. Lipofectamine 2000 dna transfection reagent protocol. Improved delivery of cas9 proteingrna complexes using. Lipofectamine 2000 transfection reagent dartmouth college. Plasmid dna, synthetic sirna, rnai plasmids shrna, mir content and storage.
Lipofectamine 2000 reagent protocol thermo fisher scientific. 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 transfection reagent is a trusted, value reagent with many years of cited use in thousands of publications and with many cell lines. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Dna lipofectamine complexes must be made in serumfree medium such as optimem reduced serum medium and can be added directly to cells in culture medium, in the presence or absence of serumantibiotic. Lipofectamine transfection reagent is a value alternative for transfection of plasmid dna into eukaryotic cells with a simplified protocol. Scaling up or down lipofectamine 3000 reagent transfections. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. 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. Invitrogen lipofectamine 3000 transfection reagent. Pdf merge combine pdf files free tool to merge pdf online. Although invitrogen recommends their own brand of culture medium for transfection, called optimem, we found no difference between this and conventional media. Adjust the amounts of components according to your tissue culture format. Transfecting sirna using lipofectamine rnaimax pin.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Follow manufacturers handbook protocol unless otherwise indicated. Gently combine tubes a and b and incubate at least 20 minutes, but can be up to several hours. The general requirements for a useful transfection reagent are that it is applied using simple, robust protocols to effect efficient transfection in a wide range of cell types and transfection formats, without excessive cytotoxicity. Scaling up or down transfections to transfect cells in different tissue culture formats, vary the amounts of lipofectamine 2000, nucleic acid, cells, and medium used in proportion. Lipofectamine 2000 from invitrogen biocompare product. Thee two most important factors in choosing transfecting reagent are. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Lipofectamine 2000 and 3000 summary in this comparative study, transfections were performed with dnain and compared to the current market leader, lipofectamine 2000 lf2k, using a series of cultured celllines and primary cells. Use this procedure to forward transfect sirna into mammalian cells in a 24well format for other formats, see scaling up or down. Superior transfection efficiency requiring lower rnai concentrations, leading to more effective gene knockdown with minimal nonspecific effects. Lipofectamine transfection reagents offer efficient delivery of dna, rna. Protocol for dna transfection and luciferase assay using pfr. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines.
The intracellular trafficking mechanism of lipofectamine. Different types of transfection reagents can be used for transfection, optimize the amounts of dna and liposomes for each type. 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. In tube b place 190 l of optimemi and 10 l lipofectamine 2000. Ka, or ikklz was performed in krascl cells using lipofectamine 2000 invitrogen. What links here related changes upload file special pages permanent link page information wikidata item cite this page. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a lipofectamine reagent.
L of dna, plus reagent and optimem combination for each transfection replicate. This reagent delivers superior transfection efficiency and improved cell viability for the widest range of hardtotransfect and common cells e. Since launching in 1993, invitrogen lipofectamine reagents have become the. Protocol generally says using 12ug dna with 68ulwell lipofectamine in 6 well plates. Lipofectamine 2000 reagent dna seed cells so they are 7090% con. Invitrogen lipofectamine 2000 transfection reagent is a proprietary. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Invitrogen lipofectamine 3000 transfection reagent 0.
Use a range of several ul of lipofectamine and ug dna to increase viability after transfection and efficiency. Invitrogen lipofectamine 3000 transfection reagent fisher. Optimization of gene transfection in murine myeloma cell. Transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. A very large number of techniques for the transfection of mammalian tissue culture cells have been described. Incubate for 5 minutes at room temperature, but not more than 10 minutes. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 3000 reagent is designed to efficiently transfect difficulttotransfect cells, yielding superior transfection performance across the broadest array of cell types. Optimemin ul, equal for dna and lipofectamine lipofectaminein ul 25. Lipofectaminez 2000 for transfection provides the following advantages.