Transient transfection of mefs transfection and transduction. Mef pkr cells produced no detectable sgs bars in b, c, and d s. I tried lipofectamine 2000 and attractene which both had a bad efficiency. The average lentiviral titers in our preparations are around 56x 10 5 x 10. I had nice results up to 60% of transfection efficiency using nih3t3. Refer to the cell lines database for a list of cell types successfully transfected. For additional information on scaling your transfection reaction, see page 4. The reagent provides high transfection efficiency in many cells types and formats. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent per well 0. For a 12 well plate, i would start with about 12ug dna and 26ul lipofectamine for each well. Viafect transfection reagent transfection assay e4981. Superior performance transit2020 outperforms fugene hd. Lipofectamine 2000 cd reagent complexes can be added directly to cells in culture medium.
Lipofectamine 2000 invitrogen, paisley, uk and complexes were prepared following the manufacturers recommended protocol. Lipofectamine 2000 transfection reagent for the transfection of nucleic acids dna and rna into. Lipofectamine rnaimax, a new transfection reagent, has been confirmed. Plate cells so they will be 7090% confluent at the time of transfection.
Is it possible to transfect cells with mrna less than. Transfection protocol lipofectamine 3000 transfection reagent. Lipofectamine 2000 transfection protocol thermo fisher. Each reaction mix is sufficient for triplicate 96well, duplicate 24well, and single well 6well transfections, and accounts for pipetting variations. Lipofectamine dna transfection reagent protocol see page 2 to view a typical plasmid transfection procedure. Dna lipofectamine 2000 cd complexes can be added directly to cells in culture medium. Dna transfection see page 2 to view a typical dna transfection procedure. Rapid and highly efficient mammalian cell engineering via. This protocol is from the kahn lab, optimized for mefs being transfected in 6well. Lipofectamine 2000 invitrogen, paisley, uk and com plexes were prepared following the manufacturers recom mended protocol. Lipofectamine 2000 cd reagent is a proprietary animal originfree formulation for transfecting nucleic acids into eukaryotic cells. Conditions were used according to the manufacturers recommendation for both lipofectamine 2000 and for jetprime. Lipofectamine messengermax reagent protocol outline a. 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 researchers use lipofectamine 2000 reagent for sirna and shrna based gene knockdown experiments as well as for gene expression studies with lipofectamine 2000 transfection reagent you ll get exceptional.
Hi, rebekka, pei is suitable for transfecting hela cells and hek293 cells, but not good for mef. If you use 24well plate format to do your experiment, the transfection protocol will be dnawell. But im not sure about the utility of this reagent for mrna transfection. What is nice about invitrogens lipofectamine 2000 is that it has an extensive, very useful manual which addresses these issues and helps tremendously cut down the initial time of protocol optimization. Lipofectamine 2000 cd reagent thermo fisher scientific. Invitrogen lipofectamine 3000 transfection reagent l3000008. Transfection efficiency was assessed by facs analysis in various cell lines 24 h after transfection in 96well plates or 24well plates. The average lentiviral titers in our preparations are around 56x 10 5 x 10 7. It is recommended to add a positive control, such as a gfp plasmid, to monitor transfection efficiency. A sample protocol is listed here for transfection experiments performed in 6well plates. Different cells require different transfection reagents.
Lipofectamine 3000 reagent thermo fisher scientific hk. 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. Mar 27, 2007 too much lipofectamine will be too cytotoxic, and too little nucleic acid will give low efficiency of transfection. Prior to use of lipofection on an alternative cell line, we strongly recommend doing. To transfect cells with sirna, follow the protocol as. Cell imaging preparation general procedure for mef cells. Transfection amounts component 96well 24well 6well dna per well 100 ng 500 ng 2500 ng plus reagent optional 0. Need help with transfecting plasmids into primary mefs. Mef conditioned medium harvested from mef culture and filtered.
Enhancement of reactive oxygen species production and. The average lentiviral titers in our preparations are around 5 x 10 6. Have you optimized the transfection condition with regard to 1 dnalipid ratios, 2 cell density, and 3 dna dosages. Transfection amounts component 96well 24well 6well final mrna per well 100 ng 500 ng 2500 ng final lipofectamine. Can anyone recommend a transfection reagent or method for. Mammalian cell transfection can be done utilizing a variety of techniques. Application note gene delivery solutions stem cell. Plates were incubated for 30 min over identical magnetic arrays that were either stationary static or moving laterally between 1 and 3 hz at 0. Component 96well 24well 6well final dna per well 50100 ng 250500 ng 1.
Highest transfection efficiency in many cell types and formats. Here we compared the transfection and viral transduction efficiency using commercially available transfection reagents including fugene 6, lipofectamine 2000 and lipofectamine 3000 in different cell lines and primary cultured cells. Lipofectamine 2000 transfection reagent life technologies, cat. Transitx2 dynamic delivery system mirus bio and lipofectamine 2000 transfection reagent thermo fisher scientific were used to transfect. Animal care and use committee iacucapproved protocol and in. Electroporation and transfection via lipofectamine 2000 l2k of. 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. Lipofectamine transfection of c2c12 cells bridges lab. Jul 17, 2007 lipofectamine 2000 is a lipid based transfection reagent for eukaryotic cells. I use 24well plates for transfections in triplicates. Lipofectamine 3000 reagent provided higher gfp transfection efficiency than lipofectamine 2000 and fugene hd reagents for all five cell lines. Most frequently we achieved best efficiency with from high to low. Optimizing transfection lipofectamine 2000 cd reagent. Comparative analysis of nonviral transfection methods in mouse.
Transfection efficiencies lower than 50% were not reported in this table. Pcr and realtime pcr, reverse transcription, cdna synthesis direct from cells. We used this kit for efficient transfection of cell lines such as hl60, mdamb231, and raw 264. Invitrogen lipofectamine 2000 is a proprietary formulation for the transfection of nucleic acids dna and rna into eukaryotic cells providing the following advantages. Feb 22, 20 lipofectamine transfection of c2c12 cells. Transfection of hek and bj cells with lipofectamine 2000. Lipofectaminea 3000 transfection reagent delivers better transfection performance than lipofectaminea 2000 reagent as demonstrated by a customer rui eduardo castro, phd, university of lisbon. Luciferase expression was compared at 24 hours post transfection using a standard luciferase assay.
Lipofectamine 2000, and lipofectamine 2000 cd x broad spectrum dna deliveryachieve high expression in many cell types, including hardtotransfect cells and insect cells x animal origin freetransit2020 provides high performance with maximum compatibility description transit2020 reagent is a new and versatile transfection solution for. The normalized gfp intensity was used as an indicator of transfection efficiency. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and reliability in a wide panel of cell lines. Cytotoxicity and transfection efficiency of lipofectamine 2000 as a. Transfection protocol lipofectamine 3000 transfection. Transitlt1 transfection reagent research reagent products. Transfection of human skin fibroblasts transfection and. To date, how to reach the maximum transfection and viral transduction efficiency is still challenging.
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. Stealthsirna transfection protocol lipofectamine 2000. The weight ratio of lipofectamine 2000 to pdna was kept constant at 3. Mouse embryonic fibroblasts mefs were obtained from the fetuses of a. To transfect cells in different tissue culture formats, vary. Preparation of cells for transfection 1624 hours before transfection plan what dnas you would like to transfect.
Comparative transfection efficiency of jetprime versus its main competitor. Transfection efficiency and protein expression using geneart crispr nuclease vector. Lipofectamine 2000 based transfection 1 seed or grow mef cells to 90% confluence e. The average lentiviral titers in our preparations are around 5 x 10. I use lipofectamine 2000 invitrogen for transfection of both sirna and plasmid dna into these 293 cell lines. However, you still need to do an optimization on these reagents. Transfection protocol lipofectamine 3000 transfection reagent mcf10a breast cancer cells complete growth medium component cat. L mrna transfection mrna can be transfected in a 24well plate with 1 or 2.
I was recently tasked with developing a crispr protocol for primary and bone. Mef cells transfected with a luciferase expression plasmid using. Lipofectamine 2000 transfection reagent from invitrogen. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. Delivery of episomal vectors into primary cells by means of. Transfection of immortalized mefs emory university. Transit2020 is a broad spectrum transfection reagent that provides superior. Lipofectamine 2000 and lipofectamine 3000 the transfection performance of cellspecific dnain reagents were compared to those of lipofectamine 2000 and lipofectamine 3000 lf3k. The transfection protocol for lipofectamine 3000 was developed to be easy to use while still ensuring optimum performance and. We utilize lipofection lipofectamine 2000 for transient gene expression.
Lipofectamine 2000 reagent protocol 202 lipofectamine 2000 dna transfection reagent protocol transfect cells according to the following chart. Lipofectamine 2000 transfection reagent for the transfection of nucleic acids dna and rna into eukaryotic cells, not necessary to remove complexes or changeadd medium after transfection, but. Plate cells in a volume of 1 ml complete growth medium per well in a 12well plate 18 24 hours before transfection 60% confluency. Do transfection in the morning following the protocol. For the following protocol, we specify volumes for a 12well plate. Enhanced transfection efficiency using lipofectamine 3000. Efficient reagent for difficult to transfect cell lines. I am trying to transfect mef cells with high transfection efficiency. The mechanism of lipofectamine 2000 mediated transmembrane. Optimization of mouse embryonic stem cell transfection for the new. A detailed protocol for mtrips assembly and characterization was described in. An improved method for increasing the efficiency of gene. We ususlly use lipofectamine ltx reagent for mef transfection, with higher efficience and lower.
The transitlt1 reagent exhibits higher expression and lower cellular toxicity compared to other transfection reagents. I compared mef, nih 3t3 and l929 cells with a lipofectamine 2000 protocol. It is not necessary to remove complexes or changeadd medium after transfection, but complexes may be removed after 46 hours. Highest transfection efficiency in many cell types and formats e. Lipofectamine 2000 transfection protocol thermo fisher bioz. I want to try to use hela cell and lipofectamine 2000 to do.
Other options will be lipofectamine 2000 invitrogen, transfectam promega. The animal originfree formulation ensures that mammalian cell culture and bioproduction processes are free of animalderived materials. Genscript recommends using lipofectamine 2000 for all transfections. Transfection guide overview of transfection methods. I tried to transfect mef with lipofectamine 2000 but i obtained low.
Improved transfection of huvec and mef cells using dna indian. The suggested amount of dna for mef cells when it is approximately 80% confluent. Protocols for transfecting the most common cell lines with. Despite the importance of transfection in mef cells, there is limited. Which transfection reagent is best for transfecting mef. 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 lipofectamine 3000 transfection reagent. Hepg2 cells were transfected with a luciferase expression plasmid using the designated reagents at the manufacturers recommended reagenttodna ratio indicated beneath each bar. Characterizing exogenous mrna delivery, trafficking, cytoplasmic. In this study, five commonly used transfection reagents, including lipofectamine 3000, lipofectamine 2000, fugene, rnaimax and lipofectin, were comprehensively analyzed in ten cell lines using a.
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