Cell Transfection Protocol

(It is usually possible to leave overnight). A dilution is prepared from the plasmid preparation DNA stock to a final concentration of 0. Mammalian cell transfection is a technique commonly used to express exogenous DNA or RNA in a host cell line (for example, for generating RNAi probes). The following protocol has been developed from the literature 1 for use in a 6-well plate with MISSION ® lentiviral. Cell transfection by calcium phosphate was one of the earliest chemical-based methods developed to deliver exogenous nucleic acids to cultured mammalian cells. Grow cells to be transfected to late-log phase in complete medium. Transient DNA Co-Transfection Protocol in a 6-Well Plate using TransIT-X2® System A. Add DNA-lipid complexes to cells. Also it allows for longer assay duration. The insertion of DNA into a cell enables the expression, or production, of proteins using the cells own machinery, whereas insertion of RNA into a cell is used to down-regulate the production of a specific protein by stopping translation. A549 Cell Line Transfection, Protocol, Kits and Reagents. Jul 12, 2013 · The aim of this work was to find a protocol ensuring high transfection efficiency in PC12 cells, while retaining viability and ability to differentiate. Gently rock plate to mix. Next day, dilute DNA in TE Buffer at 1 ug/ul. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. In addition, proteins and nanoparticles such as beads or dyes can be transfected. Protocol Note: The following protocol is written for transfection of cells in a 10cm dish. For adherent cells, plate cells at a density of 2-6 × 10 5 cells/well. Thaw cells, wash off media, add ~10-15 million cells to 20mL to 150mm. 3801 Europe +0080045738000 49622145030 www. Transfection is a process by which foreign nucleic acids are delivered into a eukaryotic cell to modify the host cell’s genetic makeup ( Kim & Eberwine, 2010; Chow et al. Protocol Outline Plate cells so they will be 70–90% confluent at the time of transfection. This unit contains two methods of calcium phosphate-based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. 25 M CaCl2 stock. Next day, if cells look badre-feed the cells with fresh complete media (10%FBS-DMEM+PSA) Cells can be harvested 48h, 72h or 96h post transfection. 4 groups were cultured for 6 hours, then washed by. (DNA concentration may be varied from 1-6 µg/ml to optimize expression, while PEI is maintained at 9 µg/ml). Wash with 1xPBS and add 0. After transfection, allow cells to grow and to express the protein for G418 resistance under non-selective conditions for about 24-48 hours (reach the peak of protein expression according to your transfection system). Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal ~70% confluency at the time of transfection. com/transfectionOptimized protocol for Lipofectamine LTX & Plus reagent:http://tools. - Important: If cells are not at the right confluence, do not wait until the next day to perform transfection, as this can significantly affect transfection efficiency. Put the trans-IT. Pass and plate the cells the day before the transfection. Split cells, placing 9ml of cells + medium in a 10cm dish. Thaw cells, wash off media, add ~10-15 million cells to 20mL to 150mm. • Transfection compatible medium enables transfection efficiencies of. 3800 fax831. You want to be sure that your fusion protein is full-length and not degraded. The cells are able to synthesize lecithin and contain a high percentage of. Transfections were performed by using INSECTOGENE according to supplier's recommendations. Cells were divided into 4 groups for transfection: negative control (siTIMP3-NC), siTIMP3-1, siTIMP3-2 and siTIMP3-3. Incubate transfection complexes at RT for 15-30 minutes. The MISSION ® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. Cloned genes can be transfected into cells for biochemical characterization, mutational analyses, investigation of the effects of gene expression on cell growth, investigation of gene regulatory. History and Development. ProcedureRequest a detailed protocol. A dilution is prepared from the plasmid preparation DNA stock to a final concentration of 0. In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. Cell seeding and Cell density at transfection. Transfection Protocol. For adherent cells, plate cells at a density of 2-6 × 10 5 cells/well. Screen wells for duplicates and mark duplicate wells. Protocol Lentiviral Transduction. Put amount of trans-IT needed into DMEM (2ml DMEM per 15cm plate). Transfection was conducted in suspension cell cultures at 37ºC in a humidified atmosphere with 5% of CO2. Each transient expression may require 1-4 × 10 7 cells, depending on the promoter. A dilution is prepared from the plasmid preparation DNA stock to a final concentration of 0. Up to 15 to 20 µg DNA can be used for one 10-cm plate. 1)Grow HEK293T cells in DMEM+10% serum. Thaw cells, wash off media, add ~10-15 million cells to 20mL to 150mm. This protocol is for transfection in a 6 cm plate. However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses. Transfection Protocols Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. For establishing a stable cell line: Split the transfected cells 24h post transfection into new 9cm dishes in the following dilutions: 1: 20; 1:10, and 1:5. Protocol Note: The following protocol is written for transfection of cells in a 10cm dish. DNA Preparation and Transfection (Day 1) 1. Prepare and add lipid-DNA complexes to cells. Depending on the time of assay, plate sparsely or more densely. This unit contains two methods of calcium phosphate-based eukaryotic cell transfection, protocols that can be used for both transient and stable transfections. 25 M CaCl2 stock. Characterize expression of the fusion protein in your cell type of interest using transient transfections (we use HeLa cells for most of our stable cell lines, and QIAGEN’s Effectene transfection reagent to give >80% transfection efficiency). Protocol supervised by Dr. Add 500 ul of BBS to this mixture and vortex. Incubate cells for 1-7 days. Typically passage cells every 2-3 days. After transfection, allow cells to grow and to express the protein for G418 resistance under non-selective conditions for about 24-48 hours (reach the peak of protein expression according to your transfection system). For each well of cells to be transfected, dilute 0. HEK-293 cells are useful for many transfection experiments, particularly the propagation of. Add DNA-lipid complexes to cells. Prepare the cells for electroporation. BBS High Efficiency Transfection Protocol Steps. To download a QIAGEN transfection protocol for your cell type, nucleic acid, and plate format of interest, visit the TransFect Protocol Database. Next day, dilute DNA in TE Buffer at 1 ug/ul. Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal ~70% confluency at the time of transfection. Once produced, lentivirus can be used for a variety of downstream. NOTE: Invitrogen Lipofectamine Transfection Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. This protocol describes the use of MISSION ® TRC shRNA Lentiviral Particles particularly for long term silencing and phenotypic observation in suspension cells. com siRNA TRANSFECTION PROTOCOL. History and Development. 3801 Europe +0080045738000 49622145030 www. This protocol can be used to produce lentivirus from a lentiviral vector transfected into Lenti-X 293T cells using a polyethyenimine (PEI) transfection protocol. Transfection P19 cells with Lipofectamine 2000 (The same protocol can be used for 293T cells) 1. TRANSIENT TRANSFECTION PROTOCOL FOR ADHERENT CELLS (FORWARD) 2. Volumes can be increased or decreased accordingly with need. FuGENE® HD Transfection Reagent can be used in transfection protocols in the presence of serum (up to 100%), allowing transfection of cell types that require continuous exposure to. Learn more at http://www. Once produced, lentivirus can be used for a variety of downstream. • 5Seed 2 x 10 cells in 500 µL growth medium for a single. Incubate ~20 min at room temperature. Optimal cell density should be reached on the day of transfection. • Transfection of cells should be performed only between passages 5 and 25 post-thaw. CHO cells are epithelial-like cells that can be grown as adherent cells or in suspension. 2) Mix 200 ug DNA with 5 ml serum-free antibiotic-free medium (we use Invitrogen Opti-MEM Cat# 31985) and vortex briefly to mix. In addition, high transfection efficiency of HEK293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. Transfection of CHO-K1 Cells Plate CHO-K1 cells at a density of 1. After transfection, allow cells to grow and to express the protein for G418 resistance under non-selective conditions for about 24-48 hours (reach the peak of protein expression according to your transfection system). All vectors and oligonucleotides were synthesized from RiboBio (Guangzhou, China), including hsa_circ_0070934 overexpression vector and small interference RNA (hsa_circ. Suggestion: Plate cells so that cell density will be ~25-50% confluent at the time of transduction. Use suspension cells directly or trypsinize adherent cells for analysis. 25 M CaCl2 stock. Protocol for HeLa cell culture and plasmid transfection AV-02HeLa cell culture 1. Drosophila S2 cells - a transfection protocol S2 cells were maintained in Schneider’s medium with 5% heat-inactivated fetal bovine serum. A sample protocol is listed here for transfection experiments performed in 6-well plates. The DharmaFECT volumes and siRNA amounts for reverse transfection are usually lower than the amounts needed for traditional transfection. Cells should be plated 18-24 hours before transfection to ensure that the cells are actively dividing and reach the appropriate cell density (generally 700% confluence) at the time-9 of transfection. Because most cultured neurons are post-mitotic - or non-dividing - cells, specialized transfection protocols are required in order to successfully induce gene expression. 1) Before transfection, cells should be ~1 x 106 cells per ml (200 ml/1L shaker flask). Transfection of cells refers to the delivery of nucleic acid (DNA or RNA) into cultured cells. Low passage, high viability cells. Prepare 293T cells the day before in 15 cm plates; 12 million cells plated the day before gives around 25 million cells at the time of transfection. The TransFect Protocol Database is an invaluable resource for transfection experiments which provides transfection protocols for specific cell types and plate formats. com/transfectionOptimized protocol for Lipofectamine LTX & Plus reagent:http://tools. If you find this doesn’t work for your specific cell type, then you can look to our cell-specific protocols below for further optimization. The Basic Protoco …. , plasmid DNA, cDNA, mRNA, miRNA, siRNA) into the cytoplasm of eukaryotic cells. This avoids having to centrifuge the cells, which can have a detrimental effect on transfection efficiency if performed shortly prior to transfection. A431 and SCL-1 cells were seeded into 6-well plates and cultured until the cell density reached 50–60%. The Transfection Protocol Library contains standard and customized user-submitted protocols for a wide variety of cells (select cell type and protocol type from the dropdown menus). History and Development. The protocol for a 24-well plate to transfect NIH3T3 cells is as follows: Plate 10,000-15,000 NIH3T3 cells per well in 0. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. Incubate cells at 37oC, 5% CO2 overnight. Cells were divided into 4 groups for transfection: negative control (siTIMP3-NC), siTIMP3-1, siTIMP3-2 and siTIMP3-3. Gently rock plate to mix. Protocol adapted from Product Manual. Higher densities can lead to spontaneous differentiation. A sample protocol is listed here for transfection experiments performed in 6-well plates. Up to 15 to 20 µg DNA can be used for one 10-cm plate. Transfection P19 cells with Lipofectamine 2000 (The same protocol can be used for 293T cells) 1. Volumes can be increased or decreased accordingly with need. For adherent cells, plate cells at a density of 2-6 × 10 5 cells/well. Low passage, high viability cells. A dilution is prepared from the plasmid preparation DNA stock to a final concentration of 0. To perform transfection experiments in other cell culture plates, simply multiply the suggested quantities by the relative surface area of your plate. (For HeLa cells, I split 1:8-1:10 for assay at 40hrs post-. The cells must be between 70-80% confluent. For the past 30 years, transfection has gained increasing popularity due to its wide application for studying cellular processes and molecular mechanisms of. 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. • Passage cells every 3–4 days to ensure that they do not enter senescence. Wash with 1xPBS and add 0. Also it allows for longer assay duration. com/content/sfs/man. Universal Transfection Reagent is used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hard-to-transfect primary cells. Transfections were performed by using INSECTOGENE according to supplier's recommendations. Once produced, lentivirus can be used for a variety of downstream. Cell passage number. , plasmid DNA, cDNA, mRNA, miRNA, siRNA) into the cytoplasm of eukaryotic cells. To obtain the highest transfection efficiency, optimize transfection conditions by varying HEK293 cell density and amount of transfection reagent. The Basic Protoco …. Learn more at http://www. Low passage, high viability cells. Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. This is often the best place to start, especially in a new cell line. Higher densities can lead to spontaneous differentiation. Use low-passage-number-cells (less than 20) or use fresh vial of cells. Most transfection kits are manufactured on base of this 'lipofection'. Put amount of trans-IT needed into DMEM (2ml DMEM per 15cm plate). Prepare plasmid DNA-lipid complexes. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids High transfection rates for adherent mammalian cell lines and primary cell cultures (suspension cells and cells from other organisms also successfully transfected but need to be immobilized) Mild treatment of cells. 5 ml tube (for 10-cm dish). • If designing an experiment that involves transfection, ensure that setup coincides with a cell passage. Cell passage number. Protocol Lentiviral Transduction. TransfeX Transfection Reagent provides high efficiency with low cell mortality. For stable transfection, nucleofection may. Split cells, placing 9ml of cells + medium in a 10cm dish. Cell-Specific Transfection Protocols NOTE: Invitrogen Lipofectamine Transfection Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. CHO cells are widely used and well-characterized in methods for cell transfection, gene amplification, and clone selection. To obtain the highest transfection efficiency, optimize transfection conditions by varying HEK293 cell density and amount of transfection reagent. Transfection protocol for suspension cells. However, if sustained gene expression is required for longer periods of time, generation of stable cell lines is a viable option. Cell transfection was performed using Lipofectamine 3000 (Invitrogen). The protocol can be scaled to produce different amounts of virus as needed. Protocol Outline Plate cells so they will be 70-90% confluent at the time of transfection. (DNA concentration may be varied from 1-6 µg/ml to optimize expression, while PEI is maintained at 9 µg/ml). Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal ~70% confluency at the time of transfection. 25 M CaCl2 stock. This het-erogeneously expressing population of resistant cells can then be used for experimental analysis. Higher densities can lead to spontaneous differentiation. To download a QIAGEN transfection protocol for your cell type, nucleic acid, and plate format of interest, visit the TransFect Protocol Database. The cells are able to synthesize lecithin and contain a high percentage of unsaturated fatty acids, which are utilized by the cytidine. This avoids having to centrifuge the cells, which can have a detrimental effect on transfection efficiency if performed shortly prior to transfection. Protocol Outline Plate cells so they will be 70–90% confluent at the time of transfection. Transfection of cells refers to the delivery of nucleic acid (DNA or RNA) into cultured cells. DNA Preparation and Transfection (Day 1) 1. Therefore, a transfection optimization should be performed for the protocol that is going to be used for subsequent experiments. com/content/sfs/man. Maintain P19 cells undifferentiated in MEM with 10% serum (7. 25 M CaCl2 stock. Next day, if cells look badre-feed the cells with fresh complete media (10%FBS-DMEM+PSA) Cells can be harvested 48h, 72h or 96h post transfection. 1)Grow HEK293T cells in DMEM+10% serum. For the transfection of primary cells and cell lines using the Nucleofector TM Technology, transfection optimization is usually not required. Incubate cells for 2-8 weeks until they become confluent. Learn more at http://www. Thaw cells, wash off media, add ~10-15 million cells to 20mL to 150mm. Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. - Important: If cells are not at the right confluence, do not wait until the next day to perform transfection, as this can significantly affect transfection efficiency. The prevelant transfection method refers to a liposome (plasmids artificially involved) transportation pathway. Volumes can be increased or decreased accordingly with need. here is the protocol for lipofectamine : Transfecting Suspension Mammalian Cells Use the following procedure to transfect mammalian cells in suspension in a 6-well format. Prepare DNA in an eppendorf by mixing together the 5 plasmids in the proportions above 3. The TransFect Protocol Database is an invaluable resource for transfection experiments which provides transfection protocols for specific cell types and plate formats. For transfection, add the expression vector plasmid DNA to the cells at a final concentration of 3 µg/ml and PEI to a final concentration of 9 µg/ml of transfection volume. Change media 2 times per week. Split cells, placing 9ml of cells + medium in a 10cm dish. Place the loosely stoppered flask in the CO₂ incubator and leave for 48 hours. Transfection Protocols. All amounts and volumes are given on a per well basis. Cell density should be 80–90% confluent on the day of transfection. Plate cells in a volume of 100 μL complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (65 – 75% confluency). 5 ml tube (for 10-cm dish). In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. 25 M CaCl2 stock. Here, we present a simple and time-saving new transfection protocol wherein cell culture plates coated with chicken egg white are seeded with suspension cells prior to transfection. Maintain P19 cells undifferentiated in MEM with 10% serum (7. Cell line-specific protocols can be found here. Protocol adapted from Product Manual. For the past 30 years, transfection has gained increasing popularity due to its wide application for studying cellular processes and molecular mechanisms of. On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. Put the trans-IT. The Transfection Protocol Library contains standard and customized user-submitted protocols for a wide variety of cells (select cell type and protocol type from the dropdown menus). The DharmaFECT volumes and siRNA amounts for reverse transfection are usually lower than the amounts needed for traditional transfection. Ensure that cells are 70 to 90% confluent at time of transfection. 5 x10 6 cells in 2 ml of For each transfection sample,. To download a QIAGEN transfection protocol for your cell type, nucleic acid, and plate format of interest, visit the TransFect Protocol Database. Plate cells in a volume of 100 μL complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (65 – 75% confluency). Protocol Lentiviral Transduction. 5 ml of complete growth medium 12-24 hours before transfection. Then I use this virus to infect my Jurkat cells for 24h and then change medium for next 1day. Expi293™ Expression System USER GUIDE For scalable transfection of Expi293F™ cells in a chemically defined, serum-free medium, using ExpiFectamine™ 293 Transfection Kit Catalog Number A14635. For adherent cells, plate cells at a density of 2-6 × 10 5 cells/well. After transfection, allow cells to grow and to express the protein for G418 resistance under non-selective conditions for about 24-48 hours (reach the peak of protein expression according to your transfection system). For transfection, add the expression vector plasmid DNA to the cells at a final concentration of 3 µg/ml and PEI to a final concentration of 9 µg/ml of transfection volume. 5 ml of fresh growth medium. As for all transfection methods, electroporation has its advantages and disadvantages. Utilize the following mix components for all experiments. Protocol supervised by Dr. In addition, high transfection efficiency of HEK293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. Grow cells to be transfected to late-log phase in complete medium. 4 groups were cultured for 6 hours, then washed by. Cell transfection was performed using Lipofectamine 3000 (Invitrogen). Drosophila S2 cells - a transfection protocol S2 cells were maintained in Schneider’s medium with 5% heat-inactivated fetal bovine serum. Each transient expression may require 1-4 × 10 7 cells, depending on the promoter. Next day, dilute DNA in TE Buffer at 1 ug/ul. Pass and plate the cells the day before the transfection. Introduction. A dilution is prepared from the plasmid preparation DNA stock to a final concentration of 0. On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. TRANSIENT TRANSFECTION PROTOCOL FOR ADHERENT CELLS (FORWARD) 2. Popular Answers (1) For transient transfection, FuGene-6 is good. Plate cells in a volume of 100 μL complete growth medium per well in a 96-well plate 18 – 24 hours before transfection (65 – 75% confluency). Add enhancers. Kenji Yamato at Tokyo Medical and Dental University Protocol 1. 3) In a second tube mix 300 ug PEI solution (1mg/ml) with 5 ml serum-free antibiotic. After transfection, allow cells to grow and to express the protein. Add 500 ul of BBS to this mixture and vortex. Protocol 2: Calculations for Transfection of 293T for Lenti/Retro Packaging. Cells must be very healthy for good transfection and virus production) DMEM+10% FBS. Transfection Reagent is tested for absence of nuclease contamination and microbial contamination. The cell number seeded should produce 40-80% confluence on the day of transfection (usually from 1:2 to 1:5). in mammalian cells. Santa Cruz Biotechnology, Inc. (DNA concentration may be varied from 1-6 µg/ml to optimize expression, while PEI is maintained at 9 µg/ml). Cells should be plated 18 to 24 hours prior to transfection so that the monolayer cell density reaches to the optimal ~70% confluency at the time of transfection. Transfection is defined as the process of inserting nucleic acids (e. Trypsinize and resuspend the HEK293T packaging cells from 2 x T-150 flasks. For adherent cells, plate cells at a density of 2-6 × 10 5 cells/well. The DharmaFECT volumes and siRNA amounts for reverse transfection are usually lower than the amounts needed for traditional transfection. This is often the best place to start, especially in a new cell line. The protocol for a 24-well transfection reaction with HEK293 cells is here: Plate 10,000-15,000 HEK293 cells per well in 0. Incubate transfection complexes at RT for 15-30 minutes. Cell-Specific Transfection Protocols NOTE: Invitrogen Lipofectamine Transfection Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Thaw a new vial of cells after 30 passages. BBS High Efficiency Transfection Protocol Steps. 5 X 10 4 cells/well. Liposome can fuse wtih the cell membrane very well and has high efficiency of transfection. Split cells, placing 9ml of cells + medium in a 10cm dish. Add enhancers. Transfection was conducted in suspension cell cultures at 37ºC in a humidified atmosphere with 5% of CO2. ExpiFectamine™ 293 Transfection Kit Protocol Outline A. Add 500 ul of BBS to this mixture and vortex. FuGENE® HD Transfection Reagent can be used in transfection protocols in the presence of serum (up to 100%), allowing transfection of cell types that require continuous exposure to. A549 cells are positive for keratin by immunoperoxidase staining. This is often the best place to start, especially in a new cell line. TRANSIENT TRANSFECTION PROTOCOL FOR ADHERENT CELLS (FORWARD) 2. Cells must be very healthy for good transfection and virus production) DMEM+10% FBS. 5 ml of fresh growth medium. Play with cell number, HBS precipitation time, plasmid concentrations and check transfection efficiency of the Phoenix cells. Wash with 1xPBS and add 0. Grow cells to be transfected to late-log phase in complete medium. The cells must be between 70-80% confluent. Increased transfection efficiency by the directed transport, especially for low amounts of nucleic acids High transfection rates for adherent mammalian cell lines and primary cell cultures (suspension cells and cells from other organisms also successfully transfected but need to be immobilized) Mild treatment of cells. In addition, high transfection efficiency of HEK293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. Cell transfection by calcium phosphate was one of the earliest chemical-based methods developed to deliver exogenous nucleic acids to cultured mammalian cells. Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Transfection protocols. Prepare DNA according to the manufacturer’s instructions for the transfection procedure. Cells must be very healthy for good transfection and virus production) DMEM+10% FBS. Next day, dilute DNA in TE Buffer at 1 ug/ul. Cell passage number. In the protocols, plasmid DNA is introduced to monolayer cell cultures via a precipitate that adheres to the cell surface. Gently rock plate to mix. Transfection Protocols Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. Transfection is the process of deliberately introducing naked or purified nucleic acids into eukaryotic cells. History and Development. Pass and plate the cells the day before the transfection. Cell-Specific Transfection Protocols NOTE: Invitrogen Lipofectamine Transfection Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. The following protocol is optimized for transient transfection of COS-7 cells in 96-well plates without pre-plating of cells 24 h prior to transfection. This avoids having to centrifuge the cells, which can have a detrimental effect on transfection efficiency if performed shortly prior to transfection. Prepare plasmid DNA-lipid complexes. CELL GROWTH: HEK293T (from CORE-T. Low passage, high viability cells. For the past 30 years, transfection has gained increasing popularity due to its wide application for studying cellular processes and molecular mechanisms of. here is the protocol for lipofectamine : Transfecting Suspension Mammalian Cells Use the following procedure to transfect mammalian cells in suspension in a 6-well format. Use low-passage-number-cells (less than 20) or use fresh vial of cells. After transfection, allow cells to grow and to express the protein for G418 resistance under non-selective conditions for about 24-48 hours (reach the peak of protein expression according to your transfection system). Up to 15 to 20 µg DNA can be used for one 10-cm plate. The transfection kit is optimized in HepG2 cells and enables a high efficiency transfer of siRNA, miRNA or plasmid DNA into the cells. For electroporation of HeLa cells, this protocol has been successful with a mixture of 2 μg of target DNA and 20-40 μg of sheared salmon sperm DNA added directly to the cuvette. 5 μg of DNA into 100 μL of Opti-MEM® I Reduced Serum Medium without. PROTOCOL: 1. First ,I transfected 4 plasmids into 293T cells and collected the supernatant after 48h. Wash with 1xPBS and add 0. The cells are able to synthesize lecithin and contain a high percentage of unsaturated fatty acids, which are utilized by the cytidine. As for all transfection methods, electroporation has its advantages and disadvantages. 5 ml of complete growth medium 12-24 hours prior to transfection. The following protocol has been developed from the literature 1 for use in a 6-well plate with MISSION ® lentiviral. Vector Protocols. ExpiFectamine™ 293 Transfection Kit Protocol Outline A. Add 500 ul of BBS to this mixture and vortex. Introduction This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine. Low passage, high viability cells. At the time of transfection, cells should be about 30-40% confluent. Protocol Outline Plate cells so they will be 70–90% confluent at the time of transfection. Add 500 ul of BBS to this mixture and vortex. For Research Use Only. CHO cells are widely used and well-characterized in methods for cell transfection, gene amplification, and clone selection. Limited Use Label License:. Gently rock plate to mix. However, the transfection efficiency is in the range of 8-12% under optimal conditions. If more than one plasmid is used for transfecting the same plate, equalize DNA amounts among different plates by adding vector. 5 ml of fresh growth medium. Maintain cell density at 80% confluence or lower. Protocol Lentiviral Transduction. This protocol can be used to produce lentivirus from a lentiviral vector transfected into Lenti-X 293T cells using a polyethyenimine (PEI) transfection protocol. If you find this doesn’t work for your specific cell type, then you can you look to our cell specific protocols for further optimization. ProcedureRequest a detailed protocol. HEK 293 cells are popular for their ease of growth and transfection (HEK293 Transfection Kit), making them a common cell culture in cancer research. This saves you the time and effort of adapting existing protocols to fit your requirements. Therefore, a transfection optimization should be performed for the protocol that is going to be used for subsequent experiments. (For HeLa cells, I split 1:8-1:10 for assay at 40hrs post-. In this article, I will review the different methods of expressing exogenous DNA or RNA. TRANSIENT TRANSFECTION PROTOCOL FOR ADHERENT CELLS (FORWARD) 2. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knockout, knockin, and transgenic mice,, , describes protocols for using electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination, and. The protocol for a 24-well plate to transfect NIH3T3 cells is as follows: Plate 10,000-15,000 NIH3T3 cells per well in 0. The transfection kit is optimized in HepG2 cells and enables a high efficiency transfer of siRNA, miRNA or plasmid DNA into the cells. Wash with 1xPBS and add 0. For each well of cells to be transfected, dilute 0. 5 ml of fresh growth medium. • The cell culture must have >90% viability and be 80% confluent on the day of transfection. Optimization of the transfection condition Generally, transfection optimization could be achieved by transfecting cells with siRNAs targeting endogenous genes such as Lamin A/C and GAPDH and then analyzing their expression by RT-PCR or Western blotting. Prepare plasmid DNA-lipid complexes. A dilution is prepared from the plasmid preparation DNA stock to a final concentration of 0. Transfection protocols often require serum-free conditions for optimal performance because serum can interfere with many commercially available transfection reagents. This unit describes electroporation of mammalian cells, including ES cells for the preparation of knockout, knockin, and transgenic mice,, , describes protocols for using electroporation in vivo to perform gene therapy for cancer therapy and DNA vaccination, and. In addition, high transfection efficiency of HEK293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. 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. Cell seeding and Cell density at transfection. 5 ml of complete growth medium 12-24 hours prior to transfection. Cell plating and transfection are performed on the same day, making this protocol rapid and convenient. Transfection. Our Universal Transfection Reagent is a unique formulation of a proprietary polymer blend. 2) Mix 200 ug DNA with 5 ml serum-free antibiotic-free medium (we use Invitrogen Opti-MEM Cat# 31985) and vortex briefly to mix. On the day of transfection, warm the PBS to 25°-37°C. Cells were divided into 4 groups for transfection: negative control (siTIMP3-NC), siTIMP3-1, siTIMP3-2 and siTIMP3-3. Incubate cell cultures overnight. The protocol for a 24-well plate to transfect NIH3T3 cells is as follows: Plate 10,000-15,000 NIH3T3 cells per well in 0. All vectors and oligonucleotides were synthesized from RiboBio (Guangzhou, China), including hsa_circ_0070934 overexpression vector and small interference RNA (hsa_circ. On the plus side, you can use it to transfect large DNA fragments and achieve good transfection efficiencies using cell lines. (It is usually possible to leave overnight). lifetechnologies. Cells must be very healthy for good transfection and virus production) DMEM+10% FBS. Transfection — the delivery of DNA or RNA into eukaryotic cells — is a powerful tool used to study and control gene expression. Incubate transfection complexes at RT for 15-30 minutes. For the past 30 years, transfection has gained increasing popularity due to its wide application for studying cellular processes and molecular mechanisms of. Protocol for stable transfection of Cl-8 or S2 cells (modified from Qiagen's SuperFect Transfection Reagent Handbook) The day before transfection the cells are split (depending on cell type) so that they are healthy and still growing. Transfection of CHO-K1 Cells Plate CHO-K1 cells at a density of 1. Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. Prepare DNA according to the manufacturer’s instructions for the transfection procedure. Cell Transfection Protocol. This saves you the time and effort of adapting existing protocols to fit your requirements. Add enhancers. The prevelant transfection method refers to a liposome (plasmids artificially involved) transportation pathway. FuGENE® HD Transfection Reagent can be used in transfection protocols in the presence of serum (up to 100%), allowing transfection of cell types that require continuous exposure to. Because most cultured neurons are post-mitotic - or non-dividing - cells, specialized transfection protocols are required in order to successfully induce gene expression. CELL GROWTH: HEK293T (from CORE-T. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells. This is often the best place to start, especially in a new cell line. We were surprised to observe that a routinely used transfection protocol, a fixed lipid/oligonucleotide ratio, resulted in variab …. Maintain P19 cells undifferentiated in MEM with 10% serum (7. Biochemistry, Molecular Biology, and Cell Biology Protocols >> Cell transfection protocol (calcium phosphate method) Based on Maniatis. HEK 293 cells are popular for their ease of growth and transfection (HEK293 Transfection Kit), making them a common cell culture in cancer research. In an effort to optimize the transfection of cell lines with antisense oligonucleotides, we examined cellular accumulation of a labeled oligonucleotide by flow cytometry. transfection protocol. However, the transfection efficiency is in the range of 8-12% under optimal conditions. A431 and SCL-1 cells were seeded into 6-well plates and cultured until the cell density reached 50–60%. For Research Use Only. lifetechnologies. Jul 12, 2013 · The aim of this work was to find a protocol ensuring high transfection efficiency in PC12 cells, while retaining viability and ability to differentiate. For the past 30 years, transfection has gained increasing popularity due to its wide application for studying cellular processes and molecular mechanisms of. This protocol can be used to produce lentivirus from a lentiviral vector transfected into Lenti-X 293T cells using a polyethyenimine (PEI) transfection protocol. Use low-passage-number-cells (less than 20) or use fresh vial of cells. Transfection of CHO-K1 Cells Plate CHO-K1 cells at a density of 1. FuGENE® HD Transfection Reagent can be used in transfection protocols in the presence of serum (up to 100%), allowing transfection of cell types that require continuous exposure to. Add 500 ul of BBS to this mixture and vortex. Optimal cell density should be reached on the day of transfection. Split cells, placing 9ml of cells + medium in a 10cm dish. For transfection, add the expression vector plasmid DNA to the cells at a final concentration of 3 µg/ml and PEI to a final concentration of 9 µg/ml of transfection volume. For each well of cells to be transfected, dilute 0. ExpiFectamine™ 293 Transfection Kit Protocol See page 7 to view a typical transfection procedure. Incubate transfection complexes at RT for 15-30 minutes. All vectors and oligonucleotides were synthesized from RiboBio (Guangzhou, China), including hsa_circ_0070934 overexpression vector and small interference RNA (hsa_circ. Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. FuGENE® HD Transfection Reagent can be used in transfection protocols in the presence of serum (up to 100%), allowing transfection of cell types that require continuous exposure to. transfection protocol. The optimized protocol for a 24-well plate to transfect A549 cells is described below: Plate 10,000-15,000 A549 cells per well in 0. Transfection. Also it allows for longer assay duration. As for all transfection methods, electroporation has its advantages and disadvantages. Our Lipofectamine Reagent Protocols have been optimized for efficiency, viability, and reproducibility across a broad range of cell types. proprietary transfection reagents are available, and the efficiency of siRNA delivery to a particular cell line will slightly differ among the reagents. For the transfection of primary cells and cell lines using the Nucleofector TM Technology, transfection optimization is usually not required. At the time of transfection, cells should be about 30-40% confluent. Target cells are compromised. For those experiments where more transfection mix is needed, simply use a multiple of the reagents described below: For cells on 24 well plates, combine equal amounts of the plasmid in question and normalization signal with L7RH-beta-Gal plasmid. Conditions ® ®. Transfection protocols. Next day, if cells look badre-feed the cells with fresh complete media (10%FBS-DMEM+PSA) Cells can be harvested 48h, 72h or 96h post transfection. Transfected cells were collected and plated on Day 0 following the plate map below; After plating, the plate was centrifuged to settle the cells in the bottom of the dish, in order to image the cells. 5 ml tube (for 10-cm dish). This protocol can be used to produce lentivirus from a lentiviral vector transfected into Lenti-X 293T cells using a polyethyenimine (PEI) transfection protocol. Seed cells in a dish so that they can double atleast twice so they can be stably transfected. Following incubation for 24 hours at 25°C, cells. Thus, transfection techniques and protocols serve as an analytical tool that facilitates the characterization of genetic functions, protein synthesis, cell growth and development. Maintain cell density at 80% confluence or lower. DNA may contain contaminants. Then I use this virus to infect my Jurkat cells for 24h and then change medium for next 1day. Next day, if cells look badre-feed the cells with fresh complete media (10%FBS-DMEM+PSA) Cells can be harvested 48h, 72h or 96h post transfection. Transfection is the process of inserting genetic material, such as DNA and double stranded RNA, into mammalian cells. Solutions 2x BBS 50 mM BES 280 mM NaCl 1. • 5Seed 2 x 10 cells in 500 µL growth medium for a single. Protocol Note: The following protocol is written for transfection of cells in a 10cm dish. The protocol indicated below is a reverse transfection protocol. For stable transfection, nucleofection may. 2) Mix 200 ug DNA with 5 ml serum-free antibiotic-free medium (we use Invitrogen Opti-MEM Cat# 31985) and vortex briefly to mix. Transient DNA Co-Transfection Protocol in a 6-Well Plate using TransIT-X2® System A. This procedure can be modified for alternative packaging cell lines or transfection reagents. Drosophila S2 cells - a transfection protocol S2 cells were maintained in Schneider's medium with 5% heat-inactivated fetal bovine serum. 25 M CaCl2 stock. com/content/sfs/man. Optimal cell density should be reached on the day of transfection. Plate cells in a volume of 100 μL complete growth medium per well in a 96-well plate 18 - 24 hours before transfection (65 - 75% confluency). Transfection of CHO-K1 Cells Plate CHO-K1 cells at a density of 1. Utilize the following mix components for all experiments. Introduction This protocol describes a general method for transfecting mammalian cells using linear polyethylenimine. Then I use. Protocol Outline Plate cells so they will be 70–90% confluent at the time of transfection. (It is usually possible to leave overnight). For high throughput siRNA screening in easy-to-transfect cells, we recommend using a reverse transfection protocol (See this reverse transfection protocol). 5 ml of complete growth medium 12-24 hours prior to transfection. Prepare plasmid DNA-lipid complexes. A dilution is prepared from the plasmid preparation DNA stock to a final concentration of 0. Santa Cruz Biotechnology, Inc. The TransFect Protocol Database is an invaluable resource for transfection experiments which provides transfection protocols for specific cell types and plate formats. Thaw cells, wash off media, add ~10-15 million cells to 20mL to 150mm. Phoenix cells should be dark blue if you use a lacZ vector such as MFG-lacZ and X-gal staining. Low passage, high viability cells. Transfection protocols. Reagent is designed for transfection of primary cells and hard-to-transfect cell lines. In addition, our Knowledge Database contains transfection data for close to 1500 cell types. Therefore, a transfection optimization should be performed for the protocol that is going to be used for subsequent experiments. ExpiFectamine™ 293 Transfection Kit Protocol See page 7 to view a typical transfection procedure. This protocol is for transfection in a 6 cm plate. Transfection protocols vary based on cell type to be transfected, transfection method, and transfection reagent. Use the table below to troubleshoot transfection experimental failure. Procedure: Warm the Opti-MEM in a 37°C water bath. • Transfection of cells should be performed only between passages 5 and 25 post-thaw. 5 ml of fresh growth medium. Tuesday Make a DNA mixture in a 15 ml tube (for 15-cm dish), or 1. However, transfection efficiency in primary cells is low, mostly due to the high mortality rates caused by the electric pulses. 25 M CaCl2 stock. Typically passage cells every 2-3 days. - Important: If cells are not at the right confluence, do not wait until the next day to perform transfection, as this can significantly affect transfection efficiency. Since the cells are allowed to grow for an additional day. (DNA concentration may be varied from 1-6 µg/ml to optimize expression, while PEI is maintained at 9 µg/ml). Phoenix cells should be dark blue if you use a lacZ vector such as MFG-lacZ and X-gal staining. This saves you the time and effort of adapting existing protocols to fit your requirements. This saves you the time and effort of adapting existing protocols to fit your requirements. For transfection, add the expression vector plasmid DNA to the cells at a final concentration of 3 µg/ml and PEI to a final concentration of 9 µg/ml of transfection volume. Transfection protocols often require serum-free conditions for optimal performance because serum can interfere with many commercially available transfection reagents. Higher densities can lead to spontaneous differentiation. This is often the best place to start, especially in a new cell line. The DharmaFECT volumes and siRNA amounts for reverse transfection are usually lower than the amounts needed for traditional transfection. FuGENE® HD Transfection Reagent can be used in transfection protocols in the presence of serum (up to 100%), allowing transfection of cell types that require continuous exposure to. In a standard transfection protocol, the cells are plated on day 1, transfected on day 2 and assayed on day 3 or 4. Use the table below to troubleshoot transfection experimental failure. The graph represents (A) pH, (B) glucose concentration, and (C) viral titers of AAV2-DJ-shGlrx-mVenus in the cell culture medium of HEK293T cells at days 3 and 5 post-transfection with viral. Cell Transfection 1 mL of each culture was placed in a 12-well tissue culture plate (Corning) for DNA transfection. However, the transfection efficiency is in the range of 8-12% under optimal conditions. Volumes can be increased or decreased accordingly with need. I've worked with 293T cells and I managed to always get good levels of transfection, without having to worry too much about cell density or reagent:DNA ratio, but with the U2OS I can't seem to be able to establish a procedure that guarantees me good transfection rates every time. Seed cells in a dish so that they can double atleast twice so they can be stably transfected. Put the trans-IT. Maintain cell density at 80% confluence or lower. For the transfection of primary cells and cell lines using the Nucleofector TM Technology, transfection optimization is usually not required. This is often the best place to start, especially in a new cell line. Volumes can be increased or decreased accordingly with need. 5 ml of complete growth medium 12-24 hours before transfection. Transfection of suspension cells has proven to be very difficult using conventional methods. BBS High Efficiency Transfection Protocol Steps. 5 μg of DNA into 100 μL of Opti-MEM® I Reduced Serum Medium without. CELL GROWTH: HEK293T (from CORE-T. Higher densities can lead to spontaneous differentiation. A sample protocol is listed here for transfection experiments performed in 6-well plates. Depending on the time of assay, plate sparsely or more densely. The cells are able to synthesize lecithin and contain a high percentage of unsaturated fatty acids, which are utilized by the cytidine. Following incubation for 24 hours at 25°C, cells. Protocol for HeLa cell culture and plasmid transfection AV-02HeLa cell culture 1. Transfection Reagent is tested for absence of nuclease contamination and microbial contamination. DNA may contain contaminants. Add 500 ul of BBS to this mixture and vortex. Protocol for stable transfection of Cl-8 or S2 cells (modified from Qiagen's SuperFect Transfection Reagent Handbook) The day before transfection the cells are split (depending on cell type) so that they are healthy and still growing. This standard protocol is referred to as forward. Use low-passage-number-cells (less than 20) or use fresh vial of cells. Cell Transfection Protocol. Transfection P19 cells with Lipofectamine 2000 (The same protocol can be used for 293T cells) Maintain P19 cells undifferentiated in MEM with 10% serum (7. 3801 Europe +0080045738000 49622145030 www. 3) In a second tube mix 300 ug PEI solution (1mg/ml) with 5 ml serum-free antibiotic. In addition, high transfection efficiency of HEK293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. The following protocol has been developed from the literature 1 for use in a 6-well plate with MISSION ® lentiviral. Cells must be very healthy for good transfection and virus production) DMEM+10% FBS. Wash with 1xPBS and add 0. Suggestion: Plate cells so that cell density will be ~25-50% confluent at the time of transduction. DNA may contain contaminants. Search for Experimental Data. The DharmaFECT volumes and siRNA amounts for reverse transfection are usually lower than the amounts needed for traditional transfection. The protocol for a 24-well transfection reaction with HEK293 cells is here: Plate 10,000-15,000 HEK293 cells per well in 0. Protocol Lentiviral Transduction. In addition, high transfection efficiency of HEK293 cells produces exogenous proteins or viruses for pharmaceutical and biomedical research purposes. Protocol for HeLa cell culture and plasmid transfection AV-02HeLa cell culture 1. CHO cells are epithelial-like cells that can be grown as adherent cells or in suspension. (DNA concentration may be varied from 1-6 µg/ml to optimize expression, while PEI is maintained at 9 µg/ml). BBS High Efficiency Transfection Protocol Steps. Prepare the cells for electroporation. Cell seeding and Cell density at transfection. The following protocol has been developed from the literature 1 for use in a 6-well plate with MISSION ® lentiviral. Protocol 4: Titering Lentiviral Vectors by Southern Blot. The graph represents (A) pH, (B) glucose concentration, and (C) viral titers of AAV2-DJ-shGlrx-mVenus in the cell culture medium of HEK293T cells at days 3 and 5 post-transfection with viral. DNA may contain contaminants. Transfection protocols. Complete culture medium with serum and antibiotics is freshly added to each well 30~60 minutes before transfection. Therefore, a transfection optimization should be performed for the protocol that is going to be used for subsequent experiments. FuGENE® HD Transfection Reagent can be used in transfection protocols in the presence of serum (up to 100%), allowing transfection of cell types that require continuous exposure to. Transfection protocols often require serum-free conditions for optimal performance because serum can interfere with many commercially available transfection reagents. It may also refer to other methods and cell types, although other terms are often preferred: "transformation" is typically used to describe non-viral DNA transfer in bacteria and non-animal eukaryotic cells, including plant cells.