Eppendorf Multiporator - Electroporation - Manual (Page 14)

38
Cell-specific application protocols are available on the Eppendorf homepage at www.eppendorf.com. The list of
applications is updated on a regular basis. (A protocol for Jurkat is included in Appendix 8.4).
If no application protocol is available for the examined cell type, the following general guidelines for the electroporation
of eukaryotic cells can be used.
To obtain the best possible transfection results, we recommend determining the optimal electroporation parameters in an
experiment. Information on how to optimize the parameters can be found in Sec. 3, "Optimizing electroporation
parameters".
4.1.1 Mycoplasma
Mycoplasma prevent efficient and reproducible electroporation of cells. Therefore it is essential to test the cells for the
presence of mycoplasma. There are several tests available.
One common method is the DNA fluorochrome staining. This test is based on DNA staining using Hoechst dye 33258 or
DAPI, which makes mycoplasma-specific DNA visible under a fluorescence microscope.
The most sensitive current method for detecting mycoplasma is by PCR. PCR detection kits are commercially available
but are more time-consuming and expensive than the aforementioned methods.
4.1.2 Cell culture
When electroporation occurs, the cells should already have passed several growth cycles. No freshly thawed or recently
transported cells should be used since this additional stress would have a negative effect on the transfection rate.
The cells should be in the exponential growth phase when transfection takes place.
4.1.3 Setting the osmolarity of the electroporation buffer
Electroporation with the Multiporator ® should ideally be carried out in hypoosmolar electroporation buffer. The tolerance
of the cells to hypoosmolar conditions has to be tested in a preliminary experiment (see Sec. 3.4).
4.1.3.1 Harvesting adherent cells
Cells should be harvested as gently as possible. According to the cell type, the following methods can be used:
• Dispase (concentration: 0.01 to 0.1 % w/v). This has proved to be the most gentle method for harvesting.
• Trypsin (HPLC-grade, without EDTA, 0.1 to 0.25 % w/v). Prior to the addition of trypsin, the cells must be washed at
least twice with PBS, without Ca
2+ and Mg 2+ .
• Scrape the cells carefully from the bottom of the culture dish.
4.1 Preparing the cells
4 Electroporation protocol
4 Electroporation protocoll
Multipor_Appli_E_poration_en.fm Seite 38 Montag, 30. Januar 2006 2:17 14