Universal Transfection Reagent Protocol

Product Number: T0956

Introduction

Our 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. This 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.

Storage

Universal Transfection Reagent can be stored at 2-8 °C for up to three months. For long-term, store at –20 °C. Allow all kit components to thaw and equilibrate to room temperature before use.

Procedure

The procedure stated below is designed for the transfection of common cell lines 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 µg/µL. Optimal amount of Universal Transfection Reagent used depends on cell type and is generally 1 – 3 µL per ug 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

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

1. To prepare cells for transfection, change the medium in each culture well. Volumes given below will transfect one well in a 6-well plate; see Table 1 for volumes using other culture dishes.
2. Tube A: to prepare DNA, add 1 µg plasmid DNA into 50 µL DMEM (high glucose, without serum). Vortex gently.
3. Tube B: to prepare transfection reagent, in a separate tube, add 3 µL Universal Transfection Reagent to 50 µL DMEM (high glucose, without serum). Vortex gently.
4. 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).
5. Add 100 µL complexes, dropwise, to the culture well (containing cells and medium). Mix with gentle swirling.

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	10	0.6	10
48-well plate	0.5	0.5	20	1.5	20
24-well plate	0.8	1	50	3	50
12-well plate	1	1.5	100	4.5	100
6-well plate	2	2	150	6	150
3.5 cm dish	2	3	100	9	100
6 cm dish	3	5	250	15	250
10 cm dish	6	8	500	24	500
T-75 flask	10	18-36	750	54-108	750

Day 2: Optional Media Change (Sensitive Cells)

A complete media change can be performed 5 - 24 hours after transfection for very sensitive cells. For most cells, we recommend the media be changed only at 48 hours post-transfection until protocol optimization requires this extra media change.

Day 4: Change Medium and Check Transfection Efficiency

1. A complete media change should be performed at 48 hours post-transfection.
2. Between 24-48 hours post-transfection, efficiency can be determined, cells collected for other applications, or stable selection started, if applicable.
   

Stationary Culture (Suspension Cells)

Day 1: Plate Cells

Plate the cells 18-24 hours before transfection. A seeding density of 0.5 - 1.0 x 10⁶/mL should be used so the cell culture plate is ready 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

1. To prepare cells for transfection, remove the medium in each culture well and replace with 0.5 mL fresh complete medium. Volumes given below will transfect one well in a 6-well plate; see Table 1 for volumes using other culture dishes.
2. Tube A: to prepare DNA, add 2 µg plasmid DNA into 100 µL DMEM (high glucose, without serum). Vortex gently.
3. Tube B: to prepare transfection reagent, in a separate tube, add 6 µL Universal Transfection Reagent to 100 µL DMEM (high glucose, without serum). Vortex gently.
4. 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).
5. Add 200 µL complexes, dropwise, to the culture well (containing cells and medium). Mix with gentle swirling.

Day 4: Change Medium and Check Transfection Efficiency

1. A complete media change should be performed at 48 hours post-transfection.
2. Between 24-48 hours post-transfection, efficiency can be determined, cells collected for other applications, or stable selection started, if applicable.