Electroporation of Eukaryotic Cells
1. Split cells 24 hours before electroporation 1:2-1:3.
2. Linearize DNA with appropriate restriction enzyme(s) (can also transfect circular DNA in transient transfections) and treat for 30 minutes with RNAase A (stock = 10 mg/ml) at
37°C. We use 10-20 ug total of DNA per sample which can either be a single plasmid or a mixture of 2-5 ug. of a plasmid containing a selectable marker (eg pSV2neo) and 10 ug of a second plasmid containing the gene of interest. All DNA is single banded in Cesium Chloride which seems to improve efficiency. It also seems to be important to remove all traces of phenol and as much RNA as possible.
3. Phenol/Chloroform x 1, Chloroform/IAA x 2 to remove all phenol which is toxic to cells.
4. Add 1/10th volume 3M NaAcetate, 2 volumes EtOH and precipitate.
5. Spin DNA and pour off ethanol in sterile hood. Wash once with 1.0 ml ‑20°C 70% EtOH and again pour off in sterile hood.
6. Let dry in sterile hood.
7. Resuspend DNA in small volume (10 ul per electroporation) sterile HeBS.
8. Spin down 107 cells per electroporation - pool and spin in 50 ml conical tube.
9. Wash once with 20 ml room temperature sterile HeBS.
10. Resuspend cells to 107 cells/0.8 ml cells in sterile room temp. HeBS and place into sterile disposable cuvette (1.2 x 107 cells/ml).
11. Add 10 ul DNA + reference DNA.
12. Shock using following settings on Biorad Gene pulser. 200-300 Volts; 960 uFd. Set capacitor to extended setting. We usually do multiple flasks at voltages spanning 200-300V. In our experience higher voltages are more efficient for cotransfection experiments using 2 or more plasmids. Note it is normal for 30-60% of the cells to die during the initial shock.
13. Let sit at room temperature 10 minutes.
14. Transfer into T-75 flask containing 20 ml non-selective medium containing 10% FCS.
15. Transfer into selection after 60 hours. We usually need to split 1:4 to 1:10 at this point or harvest cells from which to prepare extracts for CAT assay.
Solutions: 1X HeBS: (for 250 ml)
20 mM Hepes pH exactly 7.05 1.30 gm/250 ml
137 mM NaCl 2.0 gm
5 mM KCl 0.093 gm
0.7mM Na2HPO4 25 mg
6 mM Dextrose 0.270 gm
pH to 7.05 exactly
Note: Use relatively fresh HeBS. Do not keep DNA in HeBS for extended time
Reference: Chu et al. Nucl. Acids Res. 15:1311, 1987