Elizabeth D. Chao
Date: August 30, 1999
cells, has provided a powerful way to analyze the function, regulation, and interaction of
mammalian genes along with their gene products. Many methods have been developed to
facilitate the chemical and physical transfection of nucleic acids into mammalian cells,
including DEAE-dextran, calcium phosphate, liposome-mediated transfection, micro-
injection, and electroporation. The technique that we describe here utilizes electroporation,
a method which exposes the cells to a brief, defined electrical pulse to create transient pores
to allow plasmid DNA to cross the cell membrane.
(Leukemia cell line)
(Prepared under a sterile hood)
•1%1 M HEPES pH 7.4
•100 units/ml Penicillin G
•10% (v/v)Fetal Bovine Serum (GIBCO BRL)
Mix well + filter
Pre-warm to 37ºC prior to Jurkat transfection
96-well flat-bottom microtiter plates
250 ml sterile polypropylene Corning centrifuge tubes with plug seal caps
UV Sterilized electroporation cuvettes (gap width 0.4 cm)
highly purified, sterile, and free from contaminants such as endotoxins. We suggest that the
plasmid DNA should be prepared by 2X CsCl or Qiagen. Although the optimal DNA
concentration for each transfection may vary, we recommend using 2 µg of purified DNA as
a starting point.
Pre-aliquot the appropriate volumes of target plasmid and reporter into sterile microfuge
tubes. This facilitates the later step of adding the DNA directly to the cell culture, especially
when many transfections are being performed simultaneously.
Approximately 107cells should be used per Jurkat transfection. A good density for
transfection is usually 5.5 x 105cells/ml. Therefore, for a cell density of 5.5 x 105 cells/ml:
(plug-seal cap) at 1K for 5 minutes to pellet the cells.
culture media to each well in the 6-well flat-bottom multiwell Falcon plates
300 µlx (# transfections) mls to resuspend the pellet
Set the appropriate voltage (for Jurkat cells, 250 Volts)
Check the capacitance (960 µF)
Gap Width: 0.4 cm
To zap, press both buttons down and wait
Flick the cuvettes after zapping to mix contents
containing the pre-aliquoted media.
Perform the desired assay (Secreted Alkaline Phosphatase Assay used in the following
1)Berger, J., Hauber, J., Hauber R., Geiger, R., and Cullen, B.R. (1988) Gene (Amst) 66, 1-
2)Cullen, B.R., and Malim, M.H. (1992) Methods Enzymol. 216, 362-368.
cells/well in 100 µl of complete medium)
final volume of 200 µl
diethanolamine bicarbonate (pH 10.0), 1 mM methylumbelliferyl phosphate (Sigma) at
flourescent product using a Titertek Fluoroskan II (ICN) with an excitation wavelength
of 355 nm and an emission wavelength of 460 nm
Fiering, S., Northrop, J.P., Nolan, G.P., Mattila, P.S., Crabtree, G.R., and Herzenberg, L.A.
(1990) Genes & Dev. 4, 1823-1834
Resuspended in buffer A (10 mM Hepes (pH 7.8)), 15 mM KCl, 2 mM MgCl2, 1 mM
dithiothreitol, 0.1 mM EDTA
Pelleted by low speed centrifugation (Eppendorf microcentrifuge, setting 3 for 3 min)