Lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the or contact technical services for other specialized transfection protocols. Lipofectamine 2000 reagent lipofectamine ltx and plus reagents. Dilute 6 pmol rnai duplex in 100 l optimem i medium without serum in the well of the tissue culture plate. Process for an efficient lentiviral cell transduction. Fillable online lipofectamine ltx plus reagent lipofectamine. 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. The normalized gfp intensity was used as an indicator of transfection efficiency. Invitrogen lipofectamine 2000 transfection reagent is the mostcited lipidbased reagent in scienti. Transfection reagent panel target discovery institute. The intracellular trafficking mechanism of lipofectamine. Add dna lipofectamine complexes 500 ul to the 2 ml of media on the cells. Dna solution, mix gently and incubate 30 minutes at room temperature to form dna lipofectamine ltx reagent complexes. Lipofectamine ltx bioz ratings for lifescience research.
Lipofectamine ltx reagent with plus reagent from thermo. Transfection protocol lipofectamine 3000 transfection reagent. Proteins were preincubated for 20 minutes at 20 c in 100. Lipofectamine ltx reagent with plus reagent, 15338100. This makes lipofectamine ltx reagent the quickest and easiest reagent for establishing stable transfectants from challenging or refractory cell types. After 30 minutes incubation, the transfection complexes were added to the cells 75 l. The vector contained an ofp reporter gene and was transfected. The role of amyloid precursor protein in neuronal and nonneuronal.
Lipofectamine transfection reagents are the most trusted and cited. It is possible to plate and transfect the cells on the same day, but cells must adhere to the bottom of the 6well dish. Lipofectamine ltx protocol pdf lipofectamine ltx reagent offers a streamlined protocol no need to remove transfection complexes or changeadd medium following transfection. Transfection efficiency and protein expression using geneart crispr nuclease vector. Expansion of the crisprcas9 genome targeting space through.
Mix lipofectamine rnaimax gently before use, then add 1 l lipofectamine rnaimax to each. Transfection of nih3t3 cells, hela, swis 3t3, 293t with lipofectamine 2000 1. Plasmids were transfected using lipofectamine ltx reagent with plus reagent in optimem according to the manufacturers instructions 1. Enhanced and simple transfection protocol for a variety of cell lines. Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Ontarget plus smartpool sirna a pool of four target specific sirnas. Lipofectamine ltx is a cationic based transfection reagent that offers high levels of different. Pdf primary cells and certain established cells have generally been refractory.
The prepared mix is sufficient for triplicate transfections with overage. Mix gently and incubate for 515 minutes at room temperature. Add 100l of optimum to the eppendorf tube to dilute the dna and mix by tapping. With cell number and dna concentration held constant, vary the amount of lipofectamine 2000 to determine the optimal concentration usually 1. The transfection performance of cellspecific dnain reagents were compared to those of lipofectamine 2000 and lipofectamine 3000 lf3k. Mmejassisted gene knockin using talens and crisprcas9.
Lipofectamine 2000 reagent thermo fisher scientific. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. Lipofectamine ltx dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Lipofectamine ltx with plus reagent life technologies, cat. A zombie lif gene in elephants is upregulated by tp53 to induce. Invitrogenlipofectamine ltx reagent with plus reagent. Prewarm 50ml of optimum stored in cold room at 4c 10min. A new transfection reagent for effective transfection of. Gently mix tube a and b and incubate at least 20 minutes but can be several hours at room temp. Lipofectamine ltx reagent is an advanced solution for gene expression in both common and challenging cell types.
Lipofectamine ltx and plus reagents lipofectamine rnaimax freestyle max reagent 293fectin transfection reagent oligofectamine reagent dmriec optifect reagent. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Prepare transfection dilutions scaling the transfection considering the needs of the experiment. Plasmids were transfected using lipofectamine ltx reagent with plus reagent in optimem according to the manufacturers instructions. May 11, 2016 lipofectamine reagents are widely accepted as goldstandard for the safe delivery of exogenous dna or rna into cells. A lipofectamine ltx reagent, with plus reagent provides simple and convenient transfection protocol. Dna transfection with k2 transfection system in human. Incubate 5 minutes at room temperature, but not more than 10 min.
Add 100 l of the dna lipofectamine ltx complexes directly to each well containing cells and mix gently. Cells will be 7090% confluence at the time of transfection. Mar 14, 2020 lipofectamine ltx reagent is a proprietary, animalorigin free formulation for the or contact technical services for other specialized transfection protocols. Optimized protocols for over 30 different cell lines are available so much less time is needed for evaluation and optimization.
Thermo fisher lipofectamine ltx plus transfection kit lipofectamine ltx plus transfection kit, supplied by thermo fisher, used in various techniques. Thermo fisher lipofectamine ltx transfection reagent lipofectamine ltx transfection reagent, supplied by thermo fisher, used in various techniques. For each well to be transfected, prepare rnai duplex lipofectamine rnaimax complexes as follows. For several of these cells, dnain surpassed lipofectamine i i is performance figure i. Transfecting plasmid dna into hl60 cells using lipofectamine. Simple, rapid, serumcompatible protocol lipofectamine ltx reagent offers a streamlined protocol no need to remove transfection complexes or changeadd medium following transfection. Transfection protocol of lipofectamine rnaimax can be obtained from the invitrogen website. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. Forward transfection use this procedure to forward transfect sirna into mammalian cells in a 24well format for other formats, see. Transfection of nih3t3 cells, hela, swis 3t3, 293t with.
The same quantity of plasmid dna at the same ratio dnatransfection reagent 1. Jan 01, 2012 lipofectamine ltx reagent shows low cytotoxicity and therefore leads to better transfection results in many cells. At day 1 or day 9 posttransfection, cells were incubated with 3fold dilution doses of isoproterenol and luciferase activity was measured 24 hours posttreatment. Promega fugene hd and lipofectamine ltx with plus reagent for hep3b cells. Efficient and robust paramyxoviridae reverse genetics systems. Lipofectamine ltx and plus reagents lipofectamine rnaimax freestyle max reagent. This protocol is well adjusted for cancer cell lines. The lipofectamine 3000 and ltx transfection for virus production was combined with concentrating the resulting lentivirus supernatant by means of an ultrafiltration spincolumn as well as gentle pelleting of the recipient cells prior to transduction.
Genejuice transfection reagent, 293free transfection protocol pdf nanojuice. Lipofectamine ltx reagent is a plasmid transfection reagent that offers the right balance of potency and gentleness for your cells, resulting in the highest possible transfection efficiencies and viabilities. L of dna, plus reagent and optimem combination for each transfection replicate. Transfection efficiency was confirmed with egfpcoding plasmid 90%. Step tube complexation components amount per well 24well 1 tube 1 optimem i medium 25 l. Lipofectamine ltx dna transfection reagents protocol. In this protocol, we describe a procedure for crispitch v2 mediated. Invitrogen lipofectamine ltx reagent with plus reagent. Lipofectamine ltx reagent is advanced solution for gene expression in both common and challenging cell types. For comparison of t7 polymerase protein expression upon transfection of t7wt or t7opt, hek293t or bsrt75 cells were transfected in a 12well format with 0.
Biobharati life science, kolkata, india protocol using raw 264. Lipofectamine ltx plus transfection reagent thermo fisher. Enzyme transduction was performed 6 h after plating using lipofectamine ltx invitrogen, lipofectamine 3000 invitrogen and proteofectene biontex. I use 24well plates for transfections in triplicates. Despite this, a satisfactory mechanismbased explanation of their. Two complementary approaches for intracellular delivery of. Transfections were carried out using lipofectamine ltx plus gibco, life.
Combine the equal volumes of optimemdna and optimem lipofectamine. Lipofectamine transfection of c2c12 cells bridges lab protocols. Macrophages sustains innate immune functions in b homeostatic. Note, can adjust the amount of lipofectamine ltx and dna. In tube b place 3 l lipofectamine ltx in 47 l of optimemi. Lipofectamine ltx and plus regent in 100 ml of optimem were mixed, incubated for 20 min. Efficient and robust paramyxoviridae reverse genetics. Invitrogen lipofectamine ltx reagent with plus reagent 0. Efficient sirna delivery by the cationic liposome dotap in. Protocol for different transfection reagents for lentivirus package lipofectamine for 10cm dish plasmidreagent dna pgipz control 10g total dna 10g 21dr8. Reliable and e cient for workhorse and easytotransfect cell lines with a simple onetube protocol, it has been trusted by scientists since 1999. Lipofectamine ltx plus transfection reagent thermo. For satb2 and tbr1 immunostaining in p7 mice, the protocol was performed as.
To optimize the amount of lipofectamine 2000 for transfection in a 24well plate, start with cells at 90% confluency and use a fixed amount of dna 0. With cell number and dna concentration held constant, vary the amount of lipofectamine 2000 to. Add 6l of lipofectamine2000 from biostores and 100 l of. Lipofectamine transfection of c2c12 cells bridges lab. Simple protocol and efficient transfection of primary cells and macrophages.
The lipofectamine ltx and fugene hd lipidbased transfection reagents were. Many primary cell types have been successfully transfected with lipofectamine ltx reagent, including primary neural progenitor cells. For the k2 transfection system protocol, dna was resuspended in optimem medium, as follows. Lipofectamine ltx reagent, when combined with plus reagent, provides the highest level of transfection in primary cells of any lipid or polymerbased reagent. Transfection protocol lipofectamine 3000 transfection. Timeline steps procedure details day 0 1 seed cells to be 60. Lipofectamine ltx reagent with plus reagent efficiently transfects primary neural progenitor cells. No other plasmid dnaspecific transfection reagent can match the. Feb 22, 20 optimem plus required total amount of lipofectamine.
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