Eppendorf Eporator - Manual (Page 15)

Operation
Eppendorf Eporator®
English (EN)
15
• DNA concentration:The concentration of DNA can significantly influence the
transformation efficiency.
• Frequency and efficiency: The frequency is defined as the number of transformed
cells per surviving cells. The efficiency is defined as transformed cells per μg DNA. You
can obtain a high frequency by using high DNA concentrations. High efficiency is
achieved by using high cell concentration. Reducing the DNA concentration helps
prevent co-transformations of the same cell.
5.2.2 Electroporation medium
• Sensitivity of the cells: The cells are sensitive to external influences because
electroporation creates temporary pores in the cell membrane.
• Electrolysis of the medium: During electroporation, the electrolysis of the medium
significantly influences the characteristics of the medium (e.g. the pH value). Many
cells can die if fresh medium is not immediately added after electroporation for the
recovery of the cells (see Regeneration of the cells on p. 18).
• Ionic strength of the medium: The ionic strength of the medium must be taken into
account during electroporation of cells. In order to keep the resistance of the medium
as high as possible, salts must be removed from the cell and DNA preparation. Ions
remaining in the cell suspension often come from the culture medium. Higher
transformation efficiency can be obtained by removing salts from the DNA solution and
cell preparations. Generally, the lowest possible ionic solution that cells can withstand
is preferred.
5.2.3 Growth and preparation of cells
• Growth phase of cells: For optimum electroporation efficiency use bacterial (e.g. E.
coli) or yeast cells in the exponential growth phase.
• Preparation of cells: Thoroughly wash the cells in order to remove the growth medium
that affects electrocompetence.
• Concentration of cells: Use a final concentration of cells of about 1-3 x 10 11 cells/mL.
If this value is exceeded, the homogenity of the electrical field can be affected.
• Requirements for an electroporation: Each bacteria strain and yeast strain has
optimal conditions that must be determined empirically. These conditions include:
– The cell volume
– The quantity of the specific plasmid
– The used field strength (E). For E. coli, a field strength of 12-19 kV/cm is generally
required to reach a maximum transformation efficiency. The field strengths are
calculated from the voltage used and the distance of the electrodes (E = V/cm).