Universal Transfection Reagent - Lentivirus Generation in 293T Cells Protocol

Product Number: T0956

Introduction

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.

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 293T 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 3 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

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, remove the medium in each dish and replace with 5 mL fresh, complete culture medium. Volumes given below will transfect one 10 cm dish; see Table 1 for volumes using other culture dishes.
  2. Tube A: to prepare DNA, add 45 µg plasmid DNA into 500 µL DMEM (high glucose, without serum). Vortex gently.
  3. Tube B: to prepare transfection reagent, in a separate tube, add 60 µL Universal Transfection Reagent to 500 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 1 mL complexes, dropwise, to the culture dish (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)
24-well plate 0.8 6 115 8 115
12-well plate 1 12 225 15 225
6-well plate 2 23 250 30 250
3.5 cm dish 2 23 250 30 250
6 cm dish 3 23 250 30 250
10 cm dish 6 45 500 60 500


Day 2: Media Change

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

Day 4: Check Transfection Efficiency

Between 24-48 hours post-transfection, efficiency and viral titer can be determined, or cells can be collected for other applications.

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Materials

     
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