Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. The virapower and virapower ii lentiviral expression kits include the virapower lentiviral support kit plus an expression vector and the. Invitrogen lipofectamine 2000 transfection reagent 15ml. The cells were trypsinized after 48 hours, resuspended in. Highest transfection efficiency in many cell types and formats e. To ensure assembly of the ppiplatform in the nucleus, always premix your plasmids with the same amount of the platform reagent red cap before adding a transfection reagent. Lipofectamine 2000 is tested for absence of microbial contamination with blood agar plates, sabaraud dextrose agar plates, and fluid thioglycolate medium, and functionally by transfection of chok1 cells with a reporter plasmid. Lipofectamine 2000 transfection reagent dartmouth college. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may.
Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Overexpression and downregulation of proteins in vitro. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 dna transfection reagent protocol. Lipofectamine 2000 transfection reagent is a proprietary formulation. Pool dishes of cells that have been prepared for transfection. What is the appropriate way of adding lipofectamine 3000 to. For more information about lipofectamine 2000 and other transfection reagents, refer to our web site or contact technical support page. 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. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna.
Invitrogen lipofectamine 2000 transfection reagent life. Lipofectamine 2000 reagent thermo fisher scientific. Mix gently and incubate for 20 minutes at room temperature to allow the dnalipofectamine 2000 complexes to form. 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. Has anyone tried sirna transfection with lipofectamine3000. 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. Step procedure preparing cells grow your mammalian cell line of choice in the recommended medium. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Invitrogen lipofectamine 2000 transfection reagent. Refer to the cell lines database for a list of cell types successfully.
Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Life technologies corporation andor its affiliates warrant their products as set. Lipidbased transfection works in many cases and many researchers choose to use lipofectamine 2000 invitrogen according to the manufacturers instructions, especially for commonly available cell lines such as cho, 293, ht1080, hepg2, and nih 3t3. Gibco hams f12 nutrient mix 11765054 5% horse serum 1 mm gibco sodium pyruvate 160070 25 mm gibco hepes 15630106 proper culture techniques and procedures are an essential. Date 20 july 2012 description lipofectamine 2000 transfection reagent is a proprietary formulation for transfecting nucleic acids dna, rna, and. The amount of lipofectamine 2000 should be changed in proportion to the culture medium. Invitrogen lipofectamine 2000 transfection reagent 0. The cells were trypsinized after 48 hours, resuspended in 1 ml of media, and split.
Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Stealthsirna transfection protocol lipofectamine 2000. What is the appropriate way of adding lipofectamine 3000. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i. By use of this product, you accept the terms and conditions outlined in the licensing and warranty statement contained in this user manual. Lipofectamine 2000 cd reagent complexes can be added. Advanced transfection with lipofectamine 2000 reagent. Reagent composition amount virapower packaging mix contains a mixture of the plp1, plp2, and plpvsvg plasmids, 1. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. The lipofectamine 3000 is the bees knees though, i managed to get loads of transfects from a difficult to transfect cell line that had been giving us trouble with the lipofectamine 2000.
Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transient plasmid dna transfection into hela and hacat. Product is guaranteed for 6 months from the date of shipment if. Thee two most important factors in choosing transfecting reagent are. Polyethylenimine pei pei qbiogene, morgan irvine, ca and plasmid dna were each diluted with equal volumes of 150 mm nacl. To determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. In biontex experiments gfp positivity increased to 90. Valencia, ca using lipofectamine 2000 invitrogen according to manufacturers instructions. Each tube gets 8mls of optimem, 32ug of gag pol, 12.
Lipofectamine 2000 transfection protocol 10cm plates reagents. For additional information on scaling your transfection reaction, see page 4. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. After 5 minutes incubation, combine the diluted dna with the diluted lipofectamine 2000 total volume is 500. Make dna lipofectamine 3000 complexes in serumfree medium such as. Lipofectamine 2000 transfection reagent from invitrogen. Mar 03, 2014 to determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. However, after 48 hours of transfections majority of the cells. Systematic screening of commonly used commercial transfection. Invitrogen lipofectamine 2000 transfection reagent is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium. Flick each 50 ml conical tube and let sit for 5 minutes at room temperature. Plate cells so they will be 6080% confluent at the time of transfection. The reagent provides high transfection efficiency in many cells types and formats.
All amounts and volumes are given on a per well basis. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Invitrogen lipofectamine 2000 transfection reagent fisher. Effi ciency and ofp expression were analyzed 72 hours posttransfection and a u2os and b hepg2 cells showed 4fold and 80fold improvement with lipofectamine.
Use this brief procedure to transfect stealth rnai or sirna into mammalian cells in a 24well format. Biomodule transfection and control user manual unit with. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Formulation of lipofectamine 2000 and lipofectin signagen blog. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells. Add 6l of lipofectamine2000 from biostores and 100 l of. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Invitrogen transfection guide available at for a list of cell lines and. Genscript recommends using lipofectamine 2000 for all transfections.
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. Lipofectamine 2000 is commonly used for sirna transfections. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold. Optimized conditions for successful transfection of human. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. 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. Transfection protocol lipofectamine 3000 transfection. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. You can calculate this according to instructions in your reagent manual. The virapower lentiviral support kits includes the virapower packaging mix, lipofectamine 2000, and a selection agent. 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. I use 24well plates for transfections in triplicates. Lipofectamine 2000 procedure for simultaneously transfecting.
The dnalipofectamine 2000 complexes were then added to each well containing cells and medium. Types of kits this manual is supplied with the kits listed below. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Users of these products should determine if any licenses are required. Ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems.
I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. User manual corporate headquarters invitrogen corporation 1600 faraday avenue carlsbad, ca 92008 t. Lipofectamine reagents go online to view related products. Lipofectamine ltx protocol pdf lipofectamine ltx reagent offers a streamlined protocolno need to remove transfection complexes or changeadd medium following transfection. Prewarm 50ml of optimum stored in cold room at 4c 10min. Product is guaranteed for 6 months from the date of shipment if stored properly. Brightfield and dpc imaging were performed before transfection and monitored approximately every 12 hours after transfection. To maintain suitable salt concentrations for the duplex structure of your product, we recommend using the following resuspension protocol. Effects of lipofectamine 2000sirna complexes on autophagy in.