Universal Transfection Reagent - Mammalian Hard-to-Transfect Cells Protocol

Product Number: T0956


Sigma's Universal Transfection Reagent is a unique formulation of a proprietary polymer blend. Universal Transfection Reagent is used for transient and stable transfection of nucleic acids into various eukaryotic cell lines and hard-to-transfect primary cells. Fast and easy protocol is compatible with serum, serum-free medium and antibiotics. 

Reagents Provided

The Universal Transfection Reagent is provided as 1mL liquid in a single vial. This volume allows for either:

  • 40 transfections on 10 cm dishes, or
  • 65 transfections on 6 cm dishes, or
  • 110 transfections on 3.5 cm dishes.


The procedure stated below is designed for the transfection of hard-to-transfect mammalian cells with 1 µg/µl DNA plasmid (diluted in sterile molecular biology water). Culture and transfect cells in standard serum-containing or serum-free medium appropriate for the cell type.  Use good aseptic technique and use only sterile materials.

DNA plasmids should be high-quality, ethanol-precipitated, resuspended in molecular biology grade water to a final concentration of 1 ug/µL. Optimal amount of Universal Transfection Reagent used for hard-to-transfect cells is generally 4 µL per µg of plasmid DNA.

Presence of serum (>5%) and antibiotics does not inhibit transfection and in some cases increases the efficiency of transfection. Transfecting cells in the presence of serum minimizes the toxicity.

This protocol can be optimized for use with a wide variety of cell types. Seeding density, amount of DNA used, and incubation time can easily be varied to achieve higher expression and lower toxicity when needed.

Adherent Culture, transfected as suspension

Day 1: Plate Cells

Plate the cells 18-24 hours before transfection. An appropriate seeding density should be used so the cell culture plate is 90-95% confluent at the time of transfection. Serum-containing or serum-free medium appropriate for the cell type can be used for culturing the cells. 

Day 2: Transfection

  • Tube A: to prepare DNA, add 2 µg plasmid DNA into 100 µL DMEM (high glucose, without serum).  Vortex gently.  (Note: volumes given below will transfect one well in a 6-well plate; see Table 1 for volumes using other culture dishes.)
  • Tube B: to prepare transfection reagent, in a separate tube, add 8 µL Universal Transfection Reagent to 100 µL DMEM (high glucose, without serum). Vortex gently. 
  • Immediately add tube B to tube A. (Note: do not reverse the order of addition). Vortex immediately and spin down gently. Allow the complexes to form, undisturbed, at room temperature for 15-20 minutes.  (Note: do not allow complexing reaction to continue beyond 20 minutes).
  • During complexing time, prepare cells for transfection. Trypsinize cells and collect into a pellet containing 1.2 x 106 cells. Aspirate supernatant carefully. 
  • Gently resuspend the pellet in complexed solution and incubate at 37°C for 20 minutes.
  • After incubation, add 2 mL fresh, complete medium. Mix gently. Plate transfected cells into one well of a 6-well plate.

Table 1

    Tube A Tube B
Culture Dish Media Volume Per Well (mL) Total DNA per plate (µL) DMEM for diluting DNA (µL) Total Universal Transfection Reagent per plate (µL) DMEM for diluting Universal Transfection Reagent (µL)
96-well plate 0.2 0.2 20 0.8 20
48-well plate 0.5 0.5 25 2 20
24-well plate 0.8 1 50 4 50
12-well plate 1 2 100 8 100
6-well plate 2 2 100 8 100
3.5 cm dish 2 2 100 8 100
6 cm dish 3 5 250 20 250
10 cm dish 6 8 400 32 400
T-75 flask 10 36 1800 144 1800

Day 2: Media Change

A complete media change should be performed 5 - 12 hours after transfection.

Day 4: Check Transfection Efficiency

Between 24-48 hours post-transfection, efficiency can be determined, cells collected for other applications, or stable selection started, if applicable.

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