Advanced transfection with lipofectamine 2000 reagent. Transfection of fluorescently labeled oligos with lipofectamine 2000 back to table of content back to protocol and application notes. Lipofectamine 2000 procedure for simultaneously transfecting. In general, 3000 will have a better transfection efficiency 75% whereas 2000 may only be 4050%. Thee two most important factors in choosing transfecting reagent are. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. The crisprcas system is a genome editing technology that works with a cells endogenous repair mechanism. Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. C 0 lipod293 dna in vitro transfection reagent ver. Does anyone have experience with the lipofectamine 2000. Lipofectamine 2000 reagent thermo fisher scientific.
For more tips for your rnai experiment, refer to seven steps to rnai success. The controller area network can is a serial communications protocol. Transfection is the process by which nucleic acids are introduced into eukaryotic cells. 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. Does anyone have experience transfecting shsy5y cells. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Apr 24, 2012 retrovirus transfection protocol prepared by. For additional information on scaling your transfection reaction, see page 4. This comprehensive code features timetested safety concepts, structural, and fire and life safety provisions covering means of egress, interior finish. If i remember correctly the manufacturers protocols call for a 1.
Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Make sure the dna you are transfecting is relatively free of endotoxins by using an endotoxin free dna prep kit, or an endotoxin removal kit, and is free of other contaminants. Does anyone have experience with the lipofectamine 2000 toxicity. Rna lipofectamine rnaimax complexes must be made in serum free medium such as optimem. Can is a serial, multimaster, multicast protocol, which means that when the bus is free, any node can send a message multimaster, and all nodes may receive.
Lipofectamine 3000 protocol written out with timing specifics. Prepare plasmid dnalipid complexes recommend 2 doses of lipid. 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. This is the first time i will have used lipofectamine 3000, previously i only used lipofectamine 2000, and i am wondering if anyone.
The amount of lipofectamine 2000 should be changed in proportion to the culture medium. However, few studies have examined cellular responses to this delivery system. Dmem may be used to dilute lipofectaminez 2000, but transfection efficiency may be compromised. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. I use 24well plates for transfections in triplicates. Techniques vary widely and include lipidbased transfection and physical methods such as electroporation.
Make dnalipofectamine 3000 complexes in serum free medium such as optimem reduced serum medium and add directly to cells in culture. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a. I want to try to use hela cell and lipofectamine 2000 to do. Lipofectamine 2000 protocol, with 2ul of lipofectamine 2000 per well. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Select the part name and then you can download the. Lipofectamine 2000 transfection reagent dartmouth college. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Overwhelmingly more publications cite the use of lipofectamine 2000 transfection.
Pool dishes of cells that have been prepared for transfection. Add 6l of lipofectamine2000 from biostores and 100 l of. Controller area network can basics microchip technology. 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.
Overwhelmingly more publications cite the use of lipofectamine. After a 5 minute incubation in optimem, the lipofectamine 2000 and dna are combined and incubated for 20 minutes with gentle shaking at room temperature. The rest of the layers of the isoosi protocol stack are. Refer to the cell lines database for a list of cell types successfully. The transfection reactions lipoplexes were then added directly to the cells and incubated at 37. All three of these protocols specify a 5v differen tial electrical bus as the physical interface. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Exceptional transfection efficiency in a broad range of cell lines with high levels of recombinant protein expression and a simple protocol. Invitrogen lipofectamine 2000 transfection reagent. Test serum free media formulations for compatibility with lipofectaminez 2000 as some serum free formulations can inhibit cationic lipidmediated transfection. 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 transfection reagent from invitrogen. Ive used the lipofectamine 2000 transfection system to transfect human cells before. You can calculate this according to instructions in your reagent manual.
Lipofectamine 2000 is commonly used for sirna transfections. Dna complexes as described in protocol 4, steps 2 and 3. Cd293, sfm ii, vpsfm may inhibit cationic lipidmediated transfection. Page 19, table 4, columns,ubheading substitute free for face. Lipofectamine 2000 transfection reagent is a superior choice for cotransfection, given its effectiveness for transfecting both sirna and plasmid dna ideally suited for highthroughput work because transfection conditions can be easily established for automated or robotic systems. 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.
Make dna lipofectamine 3000 complexes in serum free medium such as optimem reduced serum medium and add directly to cells in culture. It can be used for transfection of adherent or suspension cells with dna or rna including stealth rnai and sirna and can be used for either transient transfections or to create stable cell lines. Both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. A highly efficient method of transfecting hep g2 cells summary.
Lipofectamine 2000 reagent protocol 202lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Sep, 2005 both lipofectamine 2000 and dna are mixed with optimem media and the quantity of each is dependant on the type of culture vessel i. Test serum free media for compatibility with lipofectamine 2000 since some serum free formulations e. I would like to transfect 293t cells with lipofectamine 3000. Jul 17, 2007 lipofectamine 2000 is designed to be used with invitrogen media with the manufacturers recommending optimem for best dnareagent complex formation. Effects of lipofectamine 2000sirna complexes on autophagy. Transfection of nih3t3 cells, hela, swis 3t3, 293t with. Despite this, a satisfactory mechanismbased explanation of their. For each well of 6well plate, dilute 2 g of dna into 100 l of serum free dmem with high glucose. Help on transfecting mcf7 and hct 116 cells protocol online. The protocol does not require removal of serum or culture medium and does not require washing or changing of medium after introducing the reagentdna complex. Download limit exceeded you have exceeded your daily download allowance. The purpose of this study is to evaluate the effect of sirna transfection using lipofectamine 2000 on cellular autophagy.
A highly efficient method of transfecting hep g2 cells. 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 reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. Lipofectamine 2000 transfection for hek293 cells about 6070% transfection rate transfect cells 6well plate. I want to try to use hela cell and lipofectamine 2000 to. Lipofectamine 3000 protocol written out with timing. The main components of the system are cas9, which is an endonuclease, and a guide rna or grna. Superior performance for cotransfection of sirna and plasmid dna with reliable performance for highthroughput applications. 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. Before seeding cells for virus package, make sure hek293t cells are in rapid replication state by growth cells to approximately 75%80% confluence in a 10 cm cell culture dish and passaging at 1.
Invitrogen lipofectamine 2000 transfection reagent fisher. Lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. 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. Invitrogen lipofectamine 2000 transfection reagent 0. The following protocol is given for transfection in 6well plates, refer to.
Lipofectamine 2000 transfection protocol 10cm plates reagents. Plate cells so they will be 7090% confluent at the time of transfection. The intracellular trafficking mechanism of lipofectamine. Thee two most important factors in choosing transfecting. The total volume of the transfection mixes was 100ul, and it was added to the medium already in the wells. As a precautionary measure it is a good idea to pass it through a sterile 0.
Lipofectamine or lipofectamine 2000 is a common transfection reagent, produced and sold by invitrogen, used in molecular and cellular biology. Does anyone have experience transfecting shsy5y cells with. Lipofectamine rnaimax transfection reagent is a proprietary formulation for transfecting small rnas e. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. Day 4 lysis i wash once with hbss ii add 100 ul passive lysis buffer iii freeze at 70 for 4 h or overnight day 5 luciferase i dual luciferase promega hope these help to design your own experiment. After 4 hours, an equal transfection volume of complete medium containing 20% serum was added, and the cells were allowed to continue incubation. Highest transfection efficiency in many cell types and formats e. Prewarm 50ml of optimum stored in cold room at 4c 10min. Other media which may be used but apparently less efficiently include dmem, rpmi 1940 and pbs. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 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.