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Transfection of siRNA or Plasmid DNA Into Primary Cells Using the Gene Pulser MXcell™ Electroporation System
Sponsored,vendor-submitted protocol   Published in November 2009 (p.19) DOI: 10.2144/000113012 Abstract

Successful electroporation of nucleic acids into primary cells requires determining optimal parameters for each cell type, a process that can be tedious. Factors that affect efficient electroporation of primary cells include waveform, pulse duration, field strength, cells and cell density, nucleic acid concentration and type, and electroporation buffer. By using a plate-based electroporation system you can easily vary many of these factors in a single experiment. The following protocol, using the Gene Pulser MXcell electroporation system, will allow you to quickly determine optimal conditions for primary and mammalian cell types.

Successful electroporation of nucleic acids into primary cells requires determining optimal parameters for each cell type, a process that can be tedious. Factors that affect efficient electroporation of primary cells include waveform, pulse duration, field strength, cells and cell density, nucleic acid concentration and type, and electroporation buffer. By using a plate-based electroporation system you can easily vary many of these factors in a single experiment. The following protocol, using the Gene Pulser MXcell electroporation system, will allow you to quickly determine optimal conditions for primary and mammalian cell types.

Materials

4 ml of cells (150 µl per well, 24 wells)

  • For fast-dividing cells (cells requiring less than 4 days to double their numbers), use 1 × 106 cells/ml of adherent cells or 2 × 106 cells/ml of cells in suspension.

  • For slow-dividing cells (cells requiring more than 4 days to double their numbers), use 2 × 106 cells/ml of adherent cells or 4 × 106 cells/ml of cells in suspension.

Appropriate growth medium PBS without calcium or magnesium 24-well tissue culture plates Nucleic acid: plasmid DNA or siRNA Gene Pulser MXcell electroporation system (Cat#165-2670)

Gene Pulser® electroporation buffer (Cat#165-2677)

96-well electroporation plate (Cat#165-2681)

Experimental Procedure Instrument Programming

Program the Gene Pulser MXcell electroporation system as described in Table 1.





Preparation of the Cells

  1. Wash the cells in PBS, pellet them, then resuspend them in 4 ml of Gene Pulser electroporation buffer to obtain the cell concentrations indicated in the Materials section.

  2. Add plasmid DNA to a final concentration of 20 µg/ml or siRNA to a final concentration of 200 nM.

  3. Pipet 150 µl of cell suspension containing plasmid DNA or siRNA into each of the 24 wells defined in Table 1.

  4. Place the lid on the plate and gently rock the plate back and forth to wet the electrodes.

  5. Place the electroporation plate securely into the plate chamber, close the lid, and press the PULSE button.

  6. Transfer the appropriate amount of cells to tissue culture plate wells and grow the cells 24–48 h until they are ready to be tested.

Evaluation of Electroporation Efficiency

Various methods, for example, fluorescence microscopy, flow cytometry, or analysis with a plate-based fluorometer, can be employed for assessing transfection efficiency when using a fluorescent marker, such as GFP or fluorescently labeled nonsilencing siRNA.

After performing one or more electroporation efficiency experiment(s), select the protocol(s) that includes the condition(s) that provided high transfection efficiency, while still maintaining cell viability.

Correspondence

Address correspondence to Bio-Rad Laboratories, Inc., Life Science Group, 2000 Alfred Nobel Drive, Hercules, CA 94547, USA; Tel.: 1-800-4BIORAD (1-800-424-6723); www.discover.bio-rad.com. For Product Technical Support: [email protected] For Literature Request: [email protected]

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