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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
  1. Prepare cells for transfection
  2. Prepare Transfectamine™ 5000-DNA mixture
  3. Add Transfectamine™ 5000-DNA mixture to cell culture
  4. Culture overnight
  5. Analyze transfection efficiency with appropriate method
Important Note

Thaw component at room temperature before starting the experiment.

PREPARATION OF WORKING SOLUTION

  1. Mix 2.5 µg of DNA with 200 µL of serum-free medium

  2. Add 7.5 µL of Transfectamine™ 5000 to Step 1

  3. 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
Component6 well plate (per well)10 cm plate
Fresh culture medium2 mL6 mL
Plasmid~2.5 µg~7.5-10 µg
Serum-free medium200 µL600 µL
Transfectamine™ 5000 Transfection Reagent~7.5 µL~22.5 µL

SAMPLE EXPERIMENTAL PROTOCOL

Preparation of Cell Culture
  1. Culture cells to ~ 90% confluency at time of transfection.
  2. 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

  1. 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
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)
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
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
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
Page updated on April 25, 2025

Ordering information

Price
Unit size
50 uL
0.5 mL
1 mL
5 mL
Catalog Number
60019600206002160022
Quantity
Add to cart

Additional ordering information

Telephone1-800-990-8053
Fax1-800-609-2943
Emailsales@aatbio.com
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Purchase orderSend to sales@aatbio.com
ShippingStandard overnight for United States, inquire for international
Request quotation

Physical properties

Solvent

Water

Storage, safety and handling

Intended useResearch Use Only (RUO)

Storage

Freeze (< -15 °C); Minimize light exposure
Transfection efficiency comparison of Transfectamine™ 5000 versus Lipofectamine 2000 reagents for CRISPR-Cas9-GFP plasmid in HeLa cells. Each reagent was used to transfect HeLa cells in a 96-well format, and the GFP expression was analyzed after 48 and 72 hours post-transfection. Transfectamine™ 5000 transfection reagent provided higher GFP transfection efficiency compared to Lipofectamine 2000.
Transfection efficiency comparison of Transfectamine™ 5000 versus Lipofectamine 2000 reagents for CRISPR-Cas9-GFP plasmid in HeLa cells. Each reagent was used to transfect HeLa cells in a 96-well format, and the GFP expression was analyzed after 48 and 72 hours post-transfection. Transfectamine™ 5000 transfection reagent provided higher GFP transfection efficiency compared to Lipofectamine 2000.
Transfection efficiency comparison of Transfectamine™ 5000 versus Lipofectamine 2000 reagents for CRISPR-Cas9-GFP plasmid in HeLa cells. Each reagent was used to transfect HeLa cells in a 96-well format, and the GFP expression was analyzed after 48 and 72 hours post-transfection. Transfectamine™ 5000 transfection reagent provided higher GFP transfection efficiency compared to Lipofectamine 2000.
Transfection efficiency comparison in HeLa cells using Transfectamine&trade; 5000, Lipofectamine 2000 and Lipofectamine 3000 reagents. Each reagent was used to transfect HeLa cells in a 96-well format, and GFP expression was analyzed 24 hours post-transfection. Transfectamine&trade; 5000 transfection reagent provided higher GFP transfection efficiency compared to Lipofectamine 2000 and Lipofectamine 3000 reagents.
Transfection efficiency comparison in CHO-K1 cells. CHO-K1 cells were cultured in 6-well plate to ~90% confluency. 2.5 ug of GFP plasmid was transfected with Lipofectamin 2000, Lipofectamine 3000 and Transfectamine&trade; 5000. Images were taken 24 hours post transfection with fluorescent microscope through FITC channel.
Cell viability comparison in CHO-K1 cells. One group of CHO K1 cells were transfected with GFP plasmid using Lipofectamine 2000, Lipofectamine 3000 and Transfectamine&trade; 5000, the second group of CHO K1 cells were treated with same amount of transfection reagent&nbsp;as the first group but without plasmid. After 48 hours, cell viability of&nbsp;each group was measured with Cell Meter&trade; Colorimetric WST-8 Cell Quantification Kit (Cat. 22770). The higher absorbance at 460nm represents more viable cells.
Transfection efficiency comparison in HeLa cells. HeLa cells were cultured in 6-well plate to 90% confluency. 2.5 &micro;g of GFP plasmid was transfect with Lipofectamine 2000, Lipofectamin 3000 and Transfectamine&trade; 5000. Signal intensity was measured by flow cytometry.

Documents

pdfProduct protocol
pdfCertificate of analysis
Transfection Reagents
Transfectamine™ 6000 CRISPR Transfection Reagent
Transfectamine™ 7000 siRNA Transfection Reagent