Megatran 1

Manufactured by OriGene

Sourced in United States

MegaTran 1.0 is a laboratory equipment designed for efficient DNA/RNA transfection. It utilizes a proprietary technology to facilitate the delivery of nucleic acids into a variety of cell types. The core function of this product is to enable researchers to effectively introduce genetic material into their cell cultures for various applications, such as gene expression studies and gene editing experiments.

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MegaTran 1.0 transfection reagent (31 citations) MegaTran (5 citations) MegaTran 1.0 reagent (5 citations) MegaTrans 1.0 (2 citations)

35 protocols using megatran 1

Generating Stable WNT7B Knockdown Cell Lines

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Three shRNA interference sequences and a control sequence were chosen (Supplementary Table 3) to construct stable WNT7B knockdown cell lines. For lentivirus production, HEK 293 T cells were plated into a 6-well plate and co-transfected with 2.5 μg shRNA lentiviral vector (pLV-shRNA-EGFP) carrying different WNT7B shRNA sequences, 1.5 μg Δ8.9 plasmid and 1 μg VSV-G plasmid using Megatran1.0 (OriGene, #TT210002) according to the manufacturer’s instructions. The supernatant was collected 48 hours and 72 hours post-transfection and filtered with 0.45 μm filters. For stable cell lines generation, SCC9 and FaDu cells were infected with different shRNA virus in 24-well plates separately, supplemented with 8 μg/ml polybrene (Solarbio, # H8761) per well, and then selected with 0.5 μg/ml puromycin (Solarbio, #P8230) for 14 days.

Chen C., Luo L., Xu C., Yang X., Liu T., Luo J., Shi W., Yang L., Zheng Y, & Yang J. (2022). Tumor specificity of WNT ligands and receptors reveals universal squamous cell carcinoma oncogenes. BMC Cancer, 22, 790.

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Cell Culture and Transfection Protocols

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HEK293T, HeLa, Panc-1 and U2OS cells were initially purchased from ATCC and cultured in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% fetal bovine serum, 100 U/ml penicilin and 100 U/ml streptomycin in 5% CO₂ at 37 °C. MEFs were isolated from female mice with Rosa26-Cre/Plk1-KI genotype at a developmental stage of 13.5 days after pregnancy and cells were cultured in Dulbecco’s modified Eagle’s medium. For plasmid DNA transfection into cells, either MegaTran1.0 (Origene) or Lipofectamine 2000 (Invitrogen) was used according to the manufacturer’s recommended protocols. G418 was used to select single positive clones that stably express transfected FLAG-Numb constructs in U2OS cells. For shRNA experiments, human Numb shRNA lentiviral particles (sc-42146-v) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA) and Numb-depleted cells were selected using puromycin as the protocol recommended. Lentivirus constructs were generated, and viral infections were performed according to previously described methods.^{31 (link)}

Shao C., Chien S.J., Farah E., Li Z., Ahmad N, & Liu X. (2017). Plk1 phosphorylation of Numb leads to impaired DNA damage response. Oncogene, 37(6), 810-820.

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Analyzing STAT1 Signaling in HeLa and U3A Cells

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HeLa cells expressing endogenous STAT1 and STAT1-negative U3A cells [26] (link) were cultured at 37°C in a humidified 5% CO₂ atmosphere in Quantum 101 medium and Dulbecco’s modified Eagle’s medium (both obtained from PAA Laboratories), respectively. For both human cell lines, media were supplemented with 10% foetal calf serum (FCS; Biochrom), 1% penicillin, and 1% streptomycin. Transfection was achieved with MegaTran1.0 (Origene) according to the manufacturer’s recommendation. Twenty-four hours after transfection, cells were either left unstimulated or stimulated with 5 ng/ml recombinant human IFNγ (Biomol). Subsequently, cells were incubated with either 500 nM staurosporine (Sigma-Aldrich) or a combination of 0.8 mM sodium vanadate and 0.2 mM H₂O₂ for the time periods indicated. The anti-fungal antibiotic leptomycin B (LMB, Sigma-Aldrich) was used at a final concentration of 10 ng/ml to block CRM1-mediated export.

Hüntelmann B., Staab J., Herrmann-Lingen C, & Meyer T. (2014). A Conserved Motif in the Linker Domain of STAT1 Transcription Factor Is Required for Both Recognition and Release from High-Affinity DNA-Binding Sites. PLoS ONE, 9(5), e97633.

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Stable ER-SFGFP-iE Cell Line Generation

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HT1080 cells were transfected with ER-SFGFP-iE using MegaTran 1.0 (OriGene, Cambridge, UK) following the manufacturer's protocol. Various dilutions (1:10 to 1:10,000) of transfected HT1080 cells were plated on 15 cm² dishes. Cells were grown in selective medium containing G418 (1 mg/ml) until colonies appeared. Single colonies were picked and transferred to 12-well plates (one colony per well) using trypsin-soaked Scienceware cloning discs (Sigma, Dorset, UK). Cells were grown until confluent and then transferred to 25 cm² flasks. The presence of ER-SFGFP-iE was confirmed by western blot using rabbit anti-GFP antibody (Santa Cruz, California, USA; cat# sc-8334, 1:200 dilution). All cell lines were regularly tested for mycoplasma infection.

Cao X., Lilla S., Cao Z., Pringle M.A., Oka O.B., Robinson P.J., Szmaja T., van Lith M., Zanivan S, & Bulleid N.J. (2020). The mammalian cytosolic thioredoxin reductase pathway acts via a membrane protein to reduce ER-localised proteins. Journal of Cell Science, 133(8), jcs241976.

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Establishment of hACE2-Expressing Cell Line

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Procedure to establish a cell line expressing human angiotensin-converting enzyme 2 (hACE2) receptor was previously described (30) and introduced in brief. HEK293T cells were used for lentiviral vector packaging and transduction. The cells were cultured in DMEM supplemented with 10% FBS (Gibco) and 1 mM nonessential amino acids (Gibco). Sub confluent HEK293T cells in 6-well plates were co-transfected with 0.72 μg of pLVX-hACE2-IRES-puro transfer plasmid, 0.64 μg of pMD2G-VSVG and 0.64 μg of pspAX.2 using transfecting reagent Megatran 1.0 (Origene).
Then, 6 h post transfection, the medium was replaced by DMEM supplemented with 3% FBS and 1 mM nonessential amino acids. Next, the lentiviral-containing supernatant was harvested at 48 h post transfection and filtered by a 0.45 μm filter (Pall). The resultant lentiviruses were used to integrate hACE2 gene into the genome of HEK293T cells. Procedure of stable lentiviral transduction was carried out as follows: HEK293T cells were seeded in a 6-well plate and transducted 24 h later with lentiviral filtrate in presence of 8 μg/mL polybrene (Macgene). Then, selection was performed under the pressure of 1 μg/mL puromycin (Invitrogen) until cells died completely. Then the cell line was verified by western blot.

Su X., Ma W., Cheng B., Wang Q., Guo Z., Zhou D., & Tang X. (2021). Efficient Inhibition of SARS-CoV-2 Using Chimeric Antisense Oligonucleotides through RNase L Activation.

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Transformation Assay for NIH 3T3 Cells

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Transformation of NIH 3T3 cells was performed following standard protocols (Clark et al., 1995 (link)). Low passage NIH 3T3 cells were plated in 60 mm dishes at a density of ~1.2 × 10⁶ cells/plate. Transfection of single plasmids (20 μg) or co-transfection of differing plasmids (10 μg each) were completed 24 hours post seeding using MegaTran 1.0 (Origene) following manufacturers protocol. 24 hours post transfection, cells were trypsinized and plated onto 60 mm dishes at a ratio of 1:4 and kept in DMEM supplemented with dexamethasone (Sigma) or G418 to determine transfection efficiency for 14 days, with media changed every 3-4 days. Cells treated with DMEM supplemented with G418 media were stained by Coomassie blue and colonies were counted to ensure equal transfection efficiency. Those plates treated with DMEM supplemented with dexamethasone were examined microscopically for signs of contact-uninhibited growth and the appearance of morphologically transformed foci. Transformed foci were counted at 10x magnification as they appeared within two 10x10 mm areas per plate. The assay was performed three times with technical duplicates of each transfection group.

Sutton M.N., Lu Z., Li Y.C., Zhou Y., Huang T., Reger A.S., Hurwitz A.M., Palzkill T., Logsdon C., Liang X., Gray J.W., Nan X., Hancock J., Wahl G.M, & Bast RC J.r. (2019). DIRAS3 (ARHI) Blocks RAS/MAPK Signaling by Binding Directly to RAS and Disrupting RAS Clusters. Cell reports, 29(11), 3448-3459.e6.

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Ras Isoform-Specific Activity Assays

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H-RAS and K-RAS specific activity assays were purchased and performed following the manufacturers protocol from CellBioLabs (STA-400-Kand STA-400-H). NIH 3T3 cells were plated in 100 mm dish at ~80% confluence. Twenty-four hours post seeding, cells were transfected with 10 μg of DNA using MegaTran 1.0 (Origene) following manufacturers protocol. 24 hours post transfection, cells were lysed as suggested following the CellBioLabs protocol and the assay was carried out according to the manufacturer’s protocol.

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Transfection of Cells with DNA and siRNA

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Cells were transfected with MegaTran 1.0 (Origene) or XtremeGeneHP (Roche) for plasmid DNA or DharmaFECT I (ThermoScientific) for siRNA, using manufacturer’s protocols. The predesigned siRNAs were purchased from Dharmacon (ThermoScientific).

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Silencing and Overexpression of HSPB1 in H9c2 Cells

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The specific siRNA against HSPB1 (siHSPB1) and scrambled siRNA were purchased from Thermo Fisher Scientific, Inc. (#315499E09, Waltham, MA, USA). The sense strand of siHSPB1 was: 5’-CCGCUGCCCAAAGCAGUCACACAAU-3’. Human pCMV-Myc-HSPB1, pCMV-HA-HSPB1, and pCMV-Myc-HSPB1-C137S plasmids were constructed by our lab and identified through complete plasmid DNA sequencing. H9c2 cells were seeded in six-well plates at a density of 1 × 10⁵ cells/well and allowed to reach approximately 50% confluence on the day of transfection. Cells were transfected with 30 pmol/well siHSPB1 using Lipofectamine RNAiMax reagent (Thermo Fisher Scientific, #13778030, Waltham, MA, USA) according to the manufacturer’s instructions. At 24 h after transfection, the cells were co-transfected with 2 μg of pCMV-Myc-HSPB1, pCMV-HA-HSPB1, or pCMV-Myc-HSPB1-C137S plasmid using MegaTran 1.0 (OriGene Technologies, #TT200004, Rockville, MD, USA) for 48h.

Wang N., Liu X., Liu K., Wang K, & Zhang H. (2023). Homo-oxidized HSPB1 protects H9c2 cells against oxidative stress via activation of KEAP1/NRF2 signaling pathway. iScience, 26(8), 107443.

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Lentiviral Transduction of Lymphoid Cells

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HEK293FT (Life Technologies) cells were cultured in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% FBS, 2 mM glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin sulfate and 1 mM sodium pyruvate (Life Technologies). Ten million cells were plated at cell on a 10 cm tissue culture dish and incubated at 37 °C 5% CO₂ incubator until the cells reach 80% confluence. The HEK293FT cells were co-transfected with either lentiviral open reading frames or shRNAs along with Lenti-vpak Packaging Kit (OriGene) using the transfection reagent MegaTran 1.0 (OriGene). After 48–72 h of transfection, the virus-containing medium was collected, spun down, filtered (0.45 µm) and used for targeting into CCRF-CEM or MOLT-4 cells by infection. The virus-infected stable clones were obtained after at least 2–3 weeks of selection in 10% FBS/RPMI-1640 with 0.5 µg/ml of puromycin (Sigma-Aldrich).

Verlekar D., Wei S.J., Cho H., Yang S, & Kang M.H. (2018). Ceramide synthase-6 confers resistance to chemotherapy by binding to CD95/Fas in T-cell acute lymphoblastic leukemia. Cell Death & Disease, 9(9), 925.

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