Escort™ IV Cotransfect siRNA with DNA

Product No. L3287

Introduction

Escort IV is a liposome suspension composed of a polycationic lipid and a neutral, non-transfecting lipid compound.  Transfection using liposomes is a commonly used method for the introduction of DNA and siRNA into eukaryotic cells. The procedure is based on the formation of a complex between the nucleic acids and the lipid reagent, which adheres to the cell surface and is taken up by the cell, presumably by endocytosis, releasing the nucleic acids into the cytoplasm.

Storage

2 – 8°C. DO NOT FREEZE.

Precautions and Disclaimer

This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Data Sheet for information regarding hazards and safe handling practices.

Procedure for transfecting adherent primary cells and cell lines

The following protocol is designed for good efficiency across a variety of cell types. For highest transfection efficiency, use the Optimization Protocol to determine the best set of conditions for your cells.

Day One: Plate Cells

Plate the cells one day prior to transfection. Use a seeding density that will provide >70% confluence about 12 hours later, generally according the chart below:

 

Culture Dish Total cells to plate (per well)
24 well plate 0.5 – 2 x105
12 well plate 0.25 - 1 x106
6 well plate or 3.5 cm dish 0.5 – 2 x106
10 cm dish 1 - 2 x106

 

Day Two: Transfection

  1. Dilute DNA and lipid reagent for complexing according to the chart below. Use only unsupplemented basal medium for dilution.  In tube A, dilute the nucleic acids in medium. In tube B, dilute the lipid reagent in medium.
  Tube A Tube B  
Culture Dish DNA volume (µL) 20µM siRNA volume (µL) Medium volume (µL) Escort IV Transfection Reagent volume (µL) Medium volume (µL) Approximate volume of media during transfection
24 well plate 0.5 2 47.5 15 335 400 µL
12 well plate 1 4 95 30 670 800 µL
6 well plate or 3.5 cm dish 2 8 190 60 1340 1.6 mL
10 cm dish 5 20 475 150 3350 4 mL

 

  1. Add tube A to tube B and mix gently. Allow complexes to form 15 minutes at room temperature.
  2. Remove half the medium from each cell culture dish and discard.
  3. Add complexes drop-wise to the cell cultures. Swirl the plates gently to distribute the complexes evenly over the cells.
  4. Incubate the cells 1.5 hours at 37°C, and then add 1ml fresh medium (use regular growth medium).  Allow the cells to continue incubating 2.5 more hours.
  5. Change the medium (use regular growth medium). Allow the cells in continue incubating 24 – 72 more hours.

Day Three and beyond: Analyze Cells

Collect and lyse the cells – they are ready to be used for other applications. Alternatively, you can passage the cells for use in other applications.

Materials

     
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