The best possible transfection results are obtained by using the original buffers from Eppendorf, which have been tested for sterility as well as for the absence of mycoplasma, endotoxins and pyrogens, and which have also under-gone a thorough cytotoxicity test.
Users may also make up this buffer themselves (see Table 1).
|Eppendorf Hypoosmolar Electroporation Buffer (PH) (no.: 4308 070.501)< TD vAlign=top align=middle>Eppendorf Isoosmolar Electroporation Buffer (PI) (no.: 4308 070.510)|
|Sterile bidistilled water||Fill up to 1000 ml||Fill up to 1000 ml|
|KCl||25 mM||25 mM|
|KH2PO4||0.3 mM||0.3 mM|
|K2HPO4||0.85 mM||0.85 mM|
|myo-Inositol*||ad 90 mOsmol/kg||ad 280 mOsmol/kg|
|pH value||7.2 0.1||7.2 0.1|
|Conductivity at 25C||3.5 mS/cm 10%||3.5 mS/cm 10%|
|Shelf-life||3 years||3 years|
*The purity of myo-Inositol may vary greatly from batch to batch. It must be pure enough to ensure that, at 280 mOsmol/kg in bidistilled water, a conductivity of 10 S/cm is not exceeded. The conductivity of individual myo-Inositol batches should be measured before the preparation of the buffer.
Ideally, electroporation should be carried out in hypoosmolar buffer, in which the cell absorbs water shortly before the pulse and then swells up as a result. A number of effects, including a decreased optimal pulse voltage ensure that the plasma membrane can be permeated more easily.
For most cell types, the 2030-minute incubation period in hypoosmolar buffer has no effect on the viability of the cells. However, the incubation in hypoosmolar buffer may induce apoptosis, or even lysis, in very sensitive cells. Therefore, it is strongly recommended to test the tolerance of the cells to hypoosmolar conditions. The easiest way of doing so is by incubating the cells for 30 minutes in hypoosmolar buffer and then performing a viability stain using trypan blue or propidium iodide. If observation under a microscope reveals lysis in more than 10% of the cells, the osmolarity of the buffer must be increased by adding isoosmolar buffer. To determine the optimal osmolarity, it is advisable to incubate the cells in different mixing ratios of hypo- and isoosmolar buffer for 30 minutes prior to the experiment (see Table 2).
This 30-minute period is the maximum incubation time for the cells in the electroporation buffer system. A new viability test followed by observation under a microscope determines the osmolarity that can be tolerated by the cells. The mixing concentrations can then be used for all subsequent experiments with this cell type.
Irrespective of the buffer system selected, it is essential to ensure that the cells do not remain in the electroporation buffer for longer than 30 minutes.
It is recommended to transfer the cells 510 minutes after the pulse into growth medium and incubate them for at least 23 hours at 37C before any centrifugation takes place. This step markedly increases the survival rate.
|Desired osmolarity||Eppendorf Hypoosmolar Buffer (ml)||Eppendorf Isoosmolar Buffer (ml)|