For hek293t cells we find that 1520 minutes is optimal. Transfection protocol lipofectamine 3000 transfection reagent. Optimum amount needed is determined from the protocol for lipofectamine 3000 transfection reagent. Genscript recommends using lipofectamine 2000 for all transfections. Collect supernatants at 48 hours post transfection and transfer polypropylene storage tube. Lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna. For detailed information about transmessenger transfection reagent, and for general guidelines. Cotransfection protocol for delivery of multiple plasmid dnas in a 6well plate with transitx2 transfection reagent. Lipofectamine 2000 transfection protocol thermo fisher bioz. Lipofectamine 2000 reagent delivers dna or sirna with excellent transfection performance for protein expression, gene silencing, and functional assays. For adherent cell lines such as hek293t, passive transfection methods using lipidbased methods e. The reagent provides high transfection efficiency in many cells types and formats. Cells that have integrated foreign dna in their genome. Scaling up or down lipofectamine 3000 reagent transfections.
I have used lipofectamine 2000 with h9c2s with a ratio of 6. F2h kit basic for analyses of proteinprotein interactions. The mechanism of lipofectamine 2000 mediated transmembrane. Co transfect your gfp and rfptagged proteins plasmid ratio 1.
Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. The amount of lipofectamine stem reagent required for optimal transfection depends on the amount of pscs plated and the amount of dna used. As described, cas9 rnps delivered into u2os cells using lipofectamine 2000 resulted in up to 80 % modification efficiency at an integrated. Jan 01, 2012 this protocol is well adjusted for cancer cell lines. Add the dna cocktail transfection buffer mix dropwise to the cells some protocols recommend waiting 1530mins for precipitate to form but i get better transfection when i dont wait 5. Lipofectamine 2000 transfection protocol thermo fisher. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Improved delivery of cas9 proteingrna complexes using. Effectene hela seed 4 x 105 cells the day before transfection and harvest cells 3648 h post transfection. Lipofectamine 2000 from invitrogen biocompare product. A11803 lipofectamine 2000 transfection reagent life technologies, cat. Use a transfection reagent appropriate for bhk cells according to manufacturers instructions. This protocol is for transfecting plasmid dna into mammalian cells.
To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. I am trying to optimize the transfection by using lipofectamine 2000 for 2000 ng of plasmid dna well with different amounts of lipofectamine 2000 5ul, 10ul in the 6well plate. Lipofectamine 2000 transfection reagent invitrogen, cat. Lipofectamine 2000 transfection reagent is a versatile transfection reagent that has been shown to effectively transfect the widest variety of adherent and suspension cell lines researchers use lipofectamine 2000 reagent for sirna and shrna based gene knockdown experiments as well as for gene expression studies with lipofectamine 2000 transfection reagent you ll get exceptional. Lipofectamine 2000 transfection reagent from invitrogen. Highest transfection efficiency in many cell types and formats. Can anyone suggest ways to optimize my cotransfection. Cells were passaged continuously with selection media for 3 months after selection for cells with the integrated. Genome engineering in hct116 cells was performed using the manufacturers transfection protocol lipofectamine 3000, thermo with media exchange at 24 hours into selection with 200. An improved method for increasing the efficiency of gene. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine. Reliable and e cient for workhorse and easytotransfect cell lines with a simple onetube protocol, it has been trusted by scientists since 1999. Rinse cells 1x w pbs, add fresh media and return to 37c make sure media is warm else cells will detach 7. To transfect cells with sirna, follow the protocol as. Lipofectamine 2000 dna transfection reagent protocol see page 2 to view a typical dna transfection procedure.
I want to try to use hela cell and lipofectamine 2000 to. As a starting point, use an amount of plasmid dna see column 4, dsrna or rnai vector dna see column 5, and lipofectamine 2000 see column 7 that falls around the midpoint of the recommended range, then optimize conditions for your cell line by varying. Jun 01, 2004 considerations for optimal transfection using lipofectamine 20002. You could easliy optimize with both vectors to find the optimal dose for expression. 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. To obtain the highest transfection efficiency and low cytotoxicity, optimize transfection conditions by varying cell density as well as dna and lipofectamine 2000 concentrations. Protocol for transfection and generation of stable cell lines using. If the starting confluence of pscs on the day of transfection is 60%, using 2 l of lipofectamine stem reagent can improve transfection efficiency. Introduction of foreign dna into the nucleus of eukaryotic cells. Lipofectamine 2000 thermo fisher scientific and 25 nm sirna according to each manufacturers protocol. Lipofectamine messengermax reagent protocol outline a. I have ordered lipofectamine 2000 from thermo fishers scientific.
The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials. I want to try to use hela cell and lipofectamine 2000 to do. For your convenience, the essential components of this protocol are now available in the gibco breast cancer starter kit. Materials puromycin dihydrochloride life technologies, cat. Transfection amounts component 96well 24well 6well final mrna per well 100 ng 500 ng 2500 ng final lipofectamine. Lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting. Component 96well 24well 6well final dna per well 100 ng 500 ng 2500 ng final lipofectamine 2000 reagent per well 0. If you own this web property, please visit our service catalog page to select a preference for handling your data otherwise you may continue to the site. To ensure assembly of the ppiplatform in the nucleus. Lipofectamine 2000 reagent can be used for transfection of plasmid dna and small rnas. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines.
Here we compared the transfection and viral transduction efficiency using commercially available transfection reagents including fugene 6, lipofectamine 2000 and lipofectamine 3000 in different cell lines and primary cultured cells. For example, for co transfection of plasmid dna and sirna in a 24well plate, we would recommend using. To date, how to reach the maximum transfection and viral transduction efficiency is still challenging. Sirna and plasmid cotransfection using lipofectamine 3000. Transfection efficiency of p19 cells is between 3050% from 5099% for many other stable cell lines. To transfect cells with sirna, follow the protocol as described for dna but. Each reagent was used to transfect hek 293, hela, lncap, a549, and hepg2 cells in a 96well format, and. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Lipofectamine 3000 transfection kit thermo fisher bioz. Make sure you remove any antibiotics from the media 24 hours before transfection and the cells need. Lipofectamine 2000 cd reagent thermo fisher scientific. H9c2 cardiomyocytes lipofectamine 2000 transfection conundrum. A sample protocol is listed here for transfection experiments performed in 6well plates. At the foundation of the popularity of this broadspectrum reagent is the reliably high efficiency and simple protocol for success across a broad range of cell lines figure 1.
I use 24well plates for transfections in triplicates. Mar 10, 2018 the protocol presented here describes the synthesis of a dendronized polymer as a highly efficient and nontoxic transfection agent. This site is scheduled to expire on march 18, 2021 dartmouth. 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.
Make sure that cells are greater than 90% confluent and vary dna. Lipofectamine 2000 transfection reagent is best transfection reagent available from life technologies for the co transfection of sirna and plasmid dna. Generally need to have 2 g of dna for each tranfection. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Evaluation of transfection methods for transient gene. Comparative transfection efficiency of jetprime versus its main competitor. Lipofectamine 2000 reagent is a proprietary formulation that facilitates highly efficient delivery of stealth rna molecules, short interfering rna sirna or.
Advanced transfection with lipofectamine 2000 reagent. C incubate at room temperature for 5min a combine the dna mixture with diluted lipofectamine 2000. Cotransfection protocol for multiple plasmid dnas mirus bio. Protocol 1 stable transfection using lipofectamine 2000 in 6 well plates. The transfection reagent lipofectamine 2000 lf2000, invitrogen is preferred for transfection. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Invitrogen lipofectamine 2000 transfection reagent is the mostcited lipidbased reagent in scienti. Lipofectamine 2000 reagent thermo fisher scientific.
It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Transfection protocol lipofectamine 3000 transfection. For some cell types, such as cos7, the transfection efficiency was over 95% while other hardertotransfect cells, such as caco2, displayed efficiencies around 75%. Transfecting a human neuroblastoma cell line with monster. However, for cotransfection of sirnachimera and plasmid dna, lipofectamine 2000 should be used. Furthermore, the optimization of the polymer as a co transfection reagent for large and multiple plasmids in cell lines is described, in addition to general considerations for co transfection experiments. The table below lists the range of recommended reagent amounts and volumes to use to transfect cells in various tissue culture formats. However, by packaging the lentiviral mirzip construct into pseudoviral particles, you can obtain highly efficient transduction and heritable expression of antimicrornaseven with most difficult to transfect cells, like primary, stem, and differentiated cells. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. The main advantage of using lipofectamine 2000 however, was its ability to transfect a range of very different cell types coupled with a high efficiency of transfection. For other types of cells such as primary, stem, or suspension cells, we suggest.
It is not necessary to change the medium, but medium may be replaced after 46 hours. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. Transfection of hek and bj cells with lipofectamine 2000. D after 20 min incubation a add the dna lipofectamine mix to the cells 1 mla 5 ml, the total medium will be 6 ml. It is best to use polypropylene instead of polystyrene tubes. Follow manufacturers handbook protocol unless otherwise indicated.
The cells were trypsinized after 48 hours, resuspended in 1 ml of media, and split. Lipofectamine 2000 also has a quick optimization protocol on the life technologies website. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 24well plates. Guidelines for cotransfection of adherent cells with sirna. Transfection of pluripotent stem cells with lipofectamine. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Cells that have incorporated the foreign dna are called transfectants. Dna is enclosed within the liposomes and the complexes fuse with the host cell membrane for dna entry into the cytoplasm and eventu ally into the nucleus. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Has anyone used lipofectamine 3000 for cotransfection of sirna and plasmid. H9c2 cardiomyocytes lipofectamine 2000 transfection. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. Lipofectamine 2000 dna transfection reagent protocol.
Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Cut plasmid with an appropriate restriction enzyme to produce a linear fragment for transfection. Carefully transfer the supernatant to a sterile polypropylene storage tube. Optimizing transfection lipofectamine 2000 cd reagent. Co transfection of plasmid dna and sirna transfect plasmid dna and sirna at the same time using lipofectamine 2000 reagent by adding 30 pmol 0. Incubate the cells at 37c in a co2 incubator for 2496 hours until you ar. To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start.
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