Mar 03, 2014 to determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Reagent composition amount virapower packaging mix contains a mixture of the plp1, plp2, and plpvsvg plasmids, 1. 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. The dnalipofectamine 2000 complexes were then added to each well containing cells and medium. Systematic screening of commonly used commercial transfection. The transfection efficacy of lipofectamine 2000 was compromised by its high toxicity, which may. All amounts and volumes are given on a per well basis. Formulation of lipofectamine 2000 and lipofectin signagen blog. You can calculate this according to instructions in your reagent manual. After 5 minutes incubation, combine the diluted dna with the diluted lipofectamine 2000 total volume is 500. Has anyone tried sirna transfection with lipofectamine3000. Combine diluted sirnachimera and lipofectamine 2000, vortex for a few second, incubated at room temperature for 20 min, then added to a 2ml culture. 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. To maintain suitable salt concentrations for the duplex structure of your product, we recommend using the following resuspension protocol.
Add 6l of lipofectamine2000 from biostores and 100 l of. 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 2000 transfection reagent. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells in a 24well format. Invitrogen transfection guide available at for a list of cell lines and.
Introduction a brief experienced users procedure for transfecting stealth rnai or sirna molecules into mammalian cells using lipofectamine 2000 reagent is described below. Each tube gets 8mls of optimem, 32ug of gag pol, 12. 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. 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.
Transfection protocol lipofectamine 3000 transfection. 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. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. Label each tube with a different viral factor oct4, sox2, klf4, or cmyc. Types of kits this manual is supplied with the kits listed below. Step procedure preparing cells grow your mammalian cell line of choice in the recommended medium. Prewarm 50ml of optimum stored in cold room at 4c 10min. What is the appropriate way of adding lipofectamine 3000 to. Valencia, ca using lipofectamine 2000 invitrogen according to manufacturers instructions. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems.
Brightfield and dpc imaging were performed before transfection and monitored approximately every 12 hours after transfection. Product is guaranteed for 6 months from the date of shipment if. Invitrogen lipofectamine 2000 transfection reagent 15ml. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. The cells were trypsinized after 48 hours, resuspended in. Biomodule transfection and control user manual unit with. Users of these products should determine if any licenses are required. 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. Optimized conditions for successful transfection of human. The cells were trypsinized after 48 hours, resuspended in 1 ml of media, and split.
Lipofectamine ltx protocol pdf lipofectamine ltx reagent offers a streamlined protocolno need to remove transfection complexes or changeadd medium following transfection. 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. Effects of lipofectamine 2000sirna complexes on autophagy in. Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Lipofectamine 2000 cd reagent complexes can be added. Make dna lipofectamine 3000 complexes in serumfree medium such as. For additional information on scaling your transfection reaction, see page 4. In biontex experiments gfp positivity increased to 90.
Transfection protocol lipofectamine 3000 transfection reagent. Dnalipofectamine 2000 complexes can be added directly to cells in culture. Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Invitrogen lipofectamine 2000 transfection reagent life. Depending on the size of culture vessel being used, the lipofectamine 2000 is diluted between 25 and 50fold. Polyethylenimine pei pei qbiogene, morgan irvine, ca and plasmid dna were each diluted with equal volumes of 150 mm nacl. Stealthsirna transfection protocol lipofectamine 2000. 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. Theres a difference between the two protocols lipofectamine 3000 has an extra reagent that needs to be added. Lipofectamine rnaimax transfection protocol see page 2 to view a typical rnaimax transfection procedure. Prior to mixing with dna, lipofectamine 2000 should be diluted in optimem i. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Lipofectamine 2000 transfection reagent is a proprietary formulation.
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 2000 transfection reagent 0. Thee two most important factors in choosing transfecting reagent are. Lipofectamine 2000 transfection protocol 10cm plates reagents. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Lipofectamine 2000 dna transfection reagent protocol. Life technologies corporation andor its affiliates warrant their products as set. User manual corporate headquarters invitrogen corporation 1600 faraday avenue carlsbad, ca 92008 t. To determine the required amount of lipofectamine 2000 for forming of lipoplexes, different amounts of lipofectamine 2000, 1, 2, 4, 6. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine 2000 transfection reagent dartmouth college. 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. The reagent provides high transfection efficiency in many cells types and formats. Mix gently and incubate for 20 minutes at room temperature to allow the dnalipofectamine 2000 complexes to form. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. I use 24well plates for transfections in triplicates. However, after 48 hours of transfections majority of the cells.
Refer to the cell lines database for a list of cell types successfully. Lipofectamine reagents go online to view related products. What is the appropriate way of adding lipofectamine 3000. In hacat cells, lipofectamine 2000 achieved a transfection efficiency of 22. Genscript recommends using lipofectamine 2000 for all transfections. Advanced transfection with lipofectamine 2000 reagent. Overexpression and downregulation of proteins in vitro.
Lipofectamine 2000 transfection reagent from invitrogen. 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. Invitrogen lipofectamine 2000 transfection reagent fisher. It is used to increase the transfection efficiency of rna including mrna and sirna or plasmid dna into in vitro cell cultures by lipofection. Pool dishes of cells that have been prepared for 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. Product is guaranteed for 6 months from the date of shipment if stored properly. Highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 reagent thermo fisher scientific. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna. Lipofectamine 2000 transfection, the expression of gfp was detected in 76,4% of hela cells with a mean fluorescence intensity of 437. Transient plasmid dna transfection into hela and hacat. Lipofectamine 2000 reagent is a lipidderived reagent designed to deliver exogenic nucleic acids dna andor rna into eukaryotic cells, either adherent or in suspension. 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. 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 is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. For more information about lipofectamine 2000 and other transfection reagents, refer to our web site or contact technical support page.
Plate cells so they will be 6080% confluent at the time of transfection. 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. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. The virapower and virapower ii lentiviral expression kits include the virapower lentiviral support kit plus an expression vector and the. The virapower lentiviral support kits includes the virapower packaging mix, lipofectamine 2000, and a selection agent. Use this brief procedure to transfect stealth rnai or sirna into mammalian cells.
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