Lipofectamine ltx reagent with plus reagent from thermo fisher. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. 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. L of dna, plus reagent and optimem combination for each transfection replicate. Lipofectamine 3000 transfection reagent leverages our most advanced lipid nanoparticle technology to provide superior transfection performance with improved application outcomes and reproducible results. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Comparison of transfection efficiency between commercialy. They have collected protocols for many cell lines and created. 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. Grow li cells transformed with pfrwt plasmid dna in appropriate volume of lb or 2xyt media with ampicillin overnight and isolate dna using standard qiagen mini or maxiprep protocol. I am not attached to invitrogen in any way but i carry 6 different cell lines and lipofectamine ltx consistently gave the highest transfection rate for all my experiments.
Pool dishes of cells that have been prepared for transfection. Prepare 6 well plates by adding bakedsterile 22x22 1. C 0 lipod293 dna in vitro transfection reagent ver. Optimizing the generation of stable neuronal cell lines. Transfection protocol lipofectamine 3000 transfection. Reduced cytotoxicity of lipofectamine ltx reagent allows the use of a greater range of lipid doses, enabling excellent transfection results despite differences in cell density, minor pipetting inaccuracies, and other variations. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Lipofectamine 2000 transfection reagent is versatile transfection reagent that has been shown to effectively transfect widest variety of adherent and suspension cell lines. Add 2 mls of media to each well and seed with 400,000 cos7 cells per coverslip. The main components of the system are cas9, which is an endonuclease, and a guide rna or grna. It is worthwhile taking a look at the invitrogen transfection website as the support offered is impressive. Lipofectamine ltx dna transfection reagent protocol. For reverse transfection b, a549 cells were plated after adding complexes following the protocol below. At approximately 45 h post transfection, cell viability i.
Cells were transfected with a firefly luciferase encoding plasmid dna 0. Metzger lab protocol book 121997 lipofectamine transfection of cho cells 1. Enhanced and simple transfection protocol for a variety of cell lines. This is lower than most transfection protocols call for. A lipofectamine ltx reagent, with plus reagent provides simple and convenient transfection protocol. If you use 24well plate format to do your experiment, the transfection protocol will be dnawell. Plate cells so they will be 7090% confluent at the time of transfection. Sep 25, 2007 lipofectamine ltx may be used with or without serum which adds to its flexibility. Researchers use lipofectamine 2000 reagent for sirna and shrnabased gene knockdown experiments, as well as for gene expression studies. Transfecting plasmid dna into nih3t3 cells using lipofectamine. This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine.
Visit technical service for other specialized transfection protocols including celltype. Invitrogen lipofectamine 3000 transfection reagent 0. Transfection should be done when dishes are 5080% confluent. Lipofectamine 2000 reagent and lipofectamine 3000 reagent were used to transfect 17 cell lines with a gfpexpressing plasmid in a 24well plate format using 0. Simple protocol and efficient transfection of primary cells and macrophages. Cas9 nuclease may be used in vivo to create targeted genome modifications. Cell medium was replaced with 2 ml optimem i, to which the mixture was added, and incubated for 4 h, after which the complexes were replaced with complete medium. Invitrogen lipofectamine ltx reagent with plus reagent 1ml. For transfecting primary cells which cannot be trypsinized, go directly to step ii, skip. Lipofectamine ltx transfection reagent from invitrogen. Optimizing the generation of stable neuronal cell lines via.
Protocol for dna transfection and luciferase assay using. The chromiumrelease assay developed in 1968 is still the most commonly used method to measure cytotoxicity by t cells and by natural killer cells. Lipofectamine ltx dna transfection reagents protocol. Lipofectamine transfection of c2c12 cells bridges lab. Laboratory 2010 lipofectamine 2000 transfection protocol 10cm plates reagents. No other plasmid dnaspecific transfection reagent can match efficiency, convenience, and gentleness of lipofectamine ltx reagent for transfection of primary, challenging, and sensitive cell lines. Lipofectamine transfection of c2c12 cells bridges lab protocols. Mix gently and incubate for 515 minutes at room temperature. Optimization of the transfection condition generally, transfection optimization could be achieved by transfecting cells with sirnas targeting endogenous genes such as lamin ac and gapdh and then analyzing their expression by rtpcr or western blotting. Since then, alternative methods have been developed using different markers of target cell. This reagent provides an advanced solution for gene expression in common cell lines, particularly chinese hampster ovary cho, and challenging cell types such. Lipofectamine transient transfection of cos7 cells to. The relative surface area of in vitro cell culture vessel and culture media volume sirna amount nmol 100 um stock exceeds tube volume 20 um stock depcdw volume ul for desired final concentration 10.
Transfection of cas9 rnp ribonucleoprotein into adherent cells using the lipofectamine rnaimax overview. The advanced protocol for hardtotransfect cells is provided only if general protocol fails to give satisfactory efficiency e. Make sufficient 10% fbs in optimem media wo antibiotics sterile filter 2. Transfection of cas9 rnp ribonucleoprotein into adherent. Refer to the cell lines database for a list of cell types successfully transfected. Lipofectamine ltx reagent is a plasmid transfection reagent that offers a balance of potency and gentleness for your cells, resulting in high transfection efficiencies and viabilities. Lipofectamine ltx may be used with or without serum which adds to its flexibility. Lipofectamine ltx with plus reagent thermo fisher scientific tw. Lipofectamine 2000 procedure for simultaneously transfecting. Lipofectamine ltx reagent is advanced solution for gene expression in both common and challenging cell types. Invitrogen lipofectamine 3000 transfection reagent from. Stroke is a lifethreatening disease that affects arteries in the brain and can be caused by the blockage of the arteries by a blood clot, resulting in seizures. Invitrogen lipofectamine 3000 transfection reagent 1.
Scaling up or down lipofectamine 3000 reagent transfections. L of lipofectamineltx was then added and allowed to complex at room temperature. They have collected protocols for many cell lines and created an excellent reference database for the tissue culture scientist. Easy protocol for dnalipofectamine ltx complex formation. Product is guaranteed for 6 months from the date of shipment if stored properly. Lipofectamine 2000 transfection reagent dartmouth college. Lipofectamine ltx was added, and the complexes were allowed to form by incubation for 25 min. Advanced protocol for transfecting hardtotransfect mammalian cells.
Lipofectamine ltx reagent is a plasmid transfection reagent that offers the right balance of potency and gentleness for your cells, resulting in the highest. Highest transfection efficiency in many cell types and formats e. 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. Make sure and evenly distribute the cells on the coverslips by swirling plate. Transfection of 293 cells in 10 cm dishes abbreviated protocol surface area of 15 cm dish is 18 x greater than that of a 6well dish. Protocol for dna transfection and luciferase assay using pfrwt plasmid. Mix gently and incubate 515 minutes at room temperature. Protocol 6 well dish plate cells and grow to 70% confluence in 2 ml media without antibiotics dmem with 10% fbs, no psg. Lipofectamine transient transfection of cos7 cells to analyze. Add the optimized volume of diluted plus reagent to diluted dna. Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the or contact technical services for other specialized transfection protocols. The crisprcas system is a genome editing technology that works with a cells endogenous repair mechanism.
Optimizing transfection of primary human umbilical vein. Target cells are loaded in vitro with radioactive chromium and lysis is determined by measuring chromium in the supernatant released by dying cells. Lipofectamine plasmid transfection bridges lab protocols. Recommended sirna resuspension volumes and concentrations table 2. Lipofectamine dna transfection reagents protocol see page 2 to view a typical plasmid transfection using a. For additional information on scaling your transfection reaction, see page 4. Volume glutamax gibco 35050 5 ml penicillinstreptomycin gibco 15140122 5 ml fbs gibco 26140079 50 ml 10% final dmem gibco 11965084 fill to 500 ml use a 0. Invitrogen lipofectamine 2000 transfection reagent.
Transfections allow for transient expression of a gene of interest in a target cell line and can be useful for short term studies of protein function. Ten cell lines were transfected with luciferaseexpressing plasmids following recommended protocols for the respective reagents. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Transfections were performed using lipofectamineltx invitrogen. This protocol results in approximately 50% to 70% cell viability, and of those viable cells, 20% to 40% are transfected when using peyfpn1. Attached at the end of this protocol is the product information sheet and protocol guidelines from gibco. Mix gently and incubate 5 minutes at room temperature.
While diluting the dna, warm lipofectamine ltx to room temperature. L of lipofectamineltx was then added and allowed to complex at room temperature for 90 min. Lipofectamine 3000 provided gentle but efficient transfection in several line of interest to our lab, heioc1 mouse cochlear 69fold, nccit and ntera2 3 fold testicular cancer lines and allowed for very nice dose response assays for cisplatin sensitvity assays. There are several ways in which to introduce cas9guide rna complexes into cells. Scaling up or down lipofectamine 3000 reagent transfections use the following table to scale the volumes for your transfection experiment. I have also had problems transfection suspension cell lines, they can be quite tricky i found that by spinning my plates downs once id added the transfection reagent i got slightly higher transfection efficiency, so this may be worth a try if you have access to an electroporator i would highly recommend going down that path.1182 7 392 977 1127 1134 756 943 1068 236 1116 1590 1147 657 1016 999 1139 1154 306 703 1071 358 335 571 1402 899 746 1168 1100 134 296 481