Eppendorf Eporator - Manual (Page 23)

Troubleshooting
Eppendorf Eporator®
English (EN)
23
6 Troubleshooting
6.1 General errors
Many factors can contribute to a low transformation efficiency:
• The set voltage: Specific voltage parameters exist for each microorganism. Some cells
die during electroporation. If the field strength is too high or too low, a low
transformation efficiency is achieved. The expected survival rate varies between 20 %
and 80 % of inserted cells. The electroporation of E. coli requires an impulse of
approximately 5 ms and field strengths between 12 kV/cm and 19 kV/cm. You should
check the transformation efficiency at different voltages in order to optimize
conditions.
Application protocols for the electroporation of various bacteria and yeast strains can
be found on the Eppendorf home page www.eppendorf.com.
• The cells: Generally, cells are transformed most efficiently when they are in an early to
medium log phase. Different growth conditions can improve the transformation
efficiency (see Growth and preparation of cells on p. 15).
If too many cells are killed, the electroporation conditions for the strain must be
optimized and the DNA preparation and cell preparation for toxic or organic
substances must be inspected (see DNA preparation on p. 14).
After electroporation, cells (especially E. coli) must be immediately transferred to a rich
medium in order to obtain good results. Even a small delay in completing this step can
lead to a significantly lower transformation efficiency (see Regeneration of the cells on
p. 18).
• The DNA: The quantity and quality of the DNA used should be checked before
electroporation. Incorrectly concentrated or degraded DNA leads to low
transformation efficiency.
Salts and other components that can have a toxic effect on cells must be removed from
the DNA preparation before the preparation process.
The DNA preparation should be added to the cells no longer than one minute before
the electroporation. DNase present in cell preparation can degrade the DNA and
thereby cause a low transformation efficiency (see DNA preparation on p. 14).
• The temperature: Electroporation cuvettes should be cooled to 0 °C - 4 °C before
electroporation (see Temperature on p. 16). This produces better results than with
electroporation cuvettes at room temperature.
If frozen cells are used, electroporation should be performed immediately after
thawing. Frozen cells can be stored a maximum of 6 - 12 months in 10 % - 15 %
glycerine at -80 °C.