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Transfectamine™ 5000 Transfection Reagent
Transfectamine™ 5000 Transfection Reagent is a powerful and versatile transfection reagent for the introduction of nucleic acids into eukaryotic cells, or more specifically, into animal cells. It can effectively transfect a variety of payloads into a variety of adherent and suspension cell lines. It can be used for plasmid DNA transfection as well as siRNA- and shRNA-based gene knockdown experiments and gene expression studies. It offers consistently high transfection efficiency in a wide variety of adherent and suspension cell lines, including difficult-to-transfect cells. The low toxicity of Transfectamine™ 5000 also allowed higher viability of transfected cells. Transfectamine™ 5000 is easier to use compare to most other transfection reagents and does not require special medium.
Example protocol
AT A GLANCE
Protocol Summary
- Prepare cells for transfection
- Prepare Transfectamine™ 5000-DNA mixture
- Add Transfectamine™ 5000-DNA mixture to cell culture
- Culture overnight
- Analyze transfection efficiency with appropriate method
Important Note
Thaw component at room temperature before starting the experiment.
PREPARATION OF WORKING SOLUTION
Mix 2.5 µg of DNA with 200 µL of serum-free medium
Add 7.5 µL of Transfectamine™ 5000 to Step 1
Mix well and incubate at room temperature for 20 minutes.
Note: Ratio of Transfectamine™ 5000 and DNA need to be optimized for different cell line, in general: Transfectamine™ 5000 Transfection Reagent (µL) to DNA (µg) Ratio = 3 - 5 µL to 1µg
Sample protocol detail for 6-well and 10 cm plate
| Component | 6 well plate (per well) | 10 cm plate |
| Fresh culture medium | 2 mL | 6 mL |
| Plasmid | ~2.5 µg | ~7.5-10 µg |
| Serum-free medium | 200 µL | 600 µL |
| Transfectamine™ 5000 Transfection Reagent | ~7.5 µL | ~22.5 µL |
SAMPLE EXPERIMENTAL PROTOCOL
Preparation of Cell Culture
- Culture cells to ~ 90% confluency at time of transfection.
- Replace with fresh growth medium before transfection. For example, replace with 2 mL of medium per well for 6-well plates and 6 mL of medium for 10 cm plates.
Transtection Protocol
Add Transfectamine™ 5000 -DNA mixture to culture plate and culture overnight.
Note: Recombinant protein can start to be detected as early as 16 hours post-transfection. Maximal expression level may be observed 72~96 hours post-transfection.
Citations
View all 48 citations: Citation Explorer
Large-scale deorphanization of Nematostella vectensis neuropeptide G protein-coupled receptors supports the independent expansion of bilaterian and cnidarian peptidergic systems
Authors: Thiel, Daniel and Guerra, Luis Alfonso Ya{\~n}ez and Kieswetter, Amanda and Cole, Alison G and Temmerman, Liesbet and Technau, Ulrich and J{\'e}kely, G{\'a}sp{\'a}r
Journal: eLife (2024): RP90674
Authors: Thiel, Daniel and Guerra, Luis Alfonso Ya{\~n}ez and Kieswetter, Amanda and Cole, Alison G and Temmerman, Liesbet and Technau, Ulrich and J{\'e}kely, G{\'a}sp{\'a}r
Journal: eLife (2024): RP90674
circ\_0008285 Regulates Glioma Progression via the miR-384/HMGB1 Axis
Authors: Yan, Manli and Hu, Caihong and Hu, Qi and Ma, Heran and Lei, Changjiang and Liu, Yamei and others,
Journal: International Journal of Genomics (2023)
Authors: Yan, Manli and Hu, Caihong and Hu, Qi and Ma, Heran and Lei, Changjiang and Liu, Yamei and others,
Journal: International Journal of Genomics (2023)
Discovery of paralogous GnRH and corazonin signaling systems in an invertebrate chordate
Authors: Yanez-Guerra, Luis Alfonso and Zandawala, Meet
Journal: bioRxiv (2023): 2023--03
Authors: Yanez-Guerra, Luis Alfonso and Zandawala, Meet
Journal: bioRxiv (2023): 2023--03
SINE Insertion in the Intron of Pig GHR May Decrease Its Expression by Acting as a Repressor
Authors: Chen Cai, and Zheng, Yao and Wang Mengli, and Murani, Eduard and D'Alessandro, Enrico and Moawad, Ali Shoaib and Wang, Xiaoyan and Wimmers, Klaus and Song, Chengyi
Journal: Animals (2021): 1871
Authors: Chen Cai, and Zheng, Yao and Wang Mengli, and Murani, Eduard and D'Alessandro, Enrico and Moawad, Ali Shoaib and Wang, Xiaoyan and Wimmers, Klaus and Song, Chengyi
Journal: Animals (2021): 1871
A Litopenaeus vannamei TRIM32 gene is involved in oxidative stress response and innate immunity
Authors: Wang, Lei and Lu, Ke-Cheng and Chen, Guo-Liang and Li, Ming and Zhang, Chao-Zheng and Chen, Yi-Hong
Journal: Fish \& Shellfish Immunology (2020): 547--555
Authors: Wang, Lei and Lu, Ke-Cheng and Chen, Guo-Liang and Li, Ming and Zhang, Chao-Zheng and Chen, Yi-Hong
Journal: Fish \& Shellfish Immunology (2020): 547--555
Page updated on September 1, 2025
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