## 1 Introduction  
Electroporation is a widely used biophysical technique that is exploited with the aim to significantly increase cell membrane permeability to exogenous biomolecules by applying external electric fields [1]. The method is routinely used for the transfer of small molecules, chemotherapeutic agents, and nucleic acids (RNA or DNA) [2; 3; 4]. The ability to effectively electroporate different types of cells (mammalian, plant, and microorganism) and transfer different types of molecules ensures the wide applicability of the method in biotechnology, clinics, and food processing [5; 6; 7].  
The electrotransfer of DNA, also known as cell electrotransfection, is a commonly exploited technique for both in vitro and in vivo applications. Even though the first experiments in DNA electrotransfer were presented more than 30 years ago, the exact mechanism of intracellular and intranuclear delivery is still under investigation [1; 8; 9]. To date, research has revealed the interdependency between DNA electrotransfer and electrophoretic forces: if the critical value of the external electric field is reached, DNA interacts with the plasma membrane exclusively at the cathode side. DNA is then translocated across the plasma membrane and transported to the nucleus for subsequent gene expression [5; 8; 10; 11; 12]. Even though pDNA electrotransfer in vitro might serve as a relatively effective technique with the selection of certain electroporation parameters and cell lines, there is much that is still unknown [7; 13]. Therefore, an understanding of the fundamental mechanisms of DNA electrotransfer--as well as knowledge regarding other important key factors that influence the efficiency of electrotransfer--is crucial and must be taken into account.  
Ca\({}^{2+}\) is one of the most important secondary messengers and is involved in a variety of different cellular processes. Therefore, the concentration of calcium ions in the cell is tightly regulated and can be significantly affected as a response to the influx of extracellular Caafter electroporation [14; 15; 16]. Changes in the number of intracellular Ca\({}^{2+}\) have been proved to be associated with ATP depletion, ROS generation, and the activation of lipases and proteases that lead to apoptotic and/or necrotic cell death [17]. It has also been suggested that Ca\({}^{2+}\) is included in the process of pDNA electrotransfection [18]. It was proposed that bivalent cations might enhance the interaction between pDNA and membrane during the application of electric pulses. However, Ca\({}^{2+}\) can also bind to polyanionic pDNA, neutralize its charge, and cause aggregation, which therefore leads to decreased transfection [18]. Thereby, the importance of the bivalent cations in the process of electrotransfection is still ambiguous.  
To date, only a few studies have analyzed the influence of Ca\({}^{2+}\) in the mechanism of DNA electrotransfer; these mainly focused on higher (>1 mM) CaCl\({}_{2}\) concentrations [19; 20]. In this study, we investigated the influence of low concentrations (0-1 mM) of Ca\({}^{2+}\) on DNA electrotransfer efficiency. Our research demonstrated the significant impact of small amounts of Ca\({}^{2+}\) on the efficiency of DNA electrotransfer and cell viability in a concentration-dependent manner. In addition, we explicitly demonstrated that Ca\({}^{2+}\) in a range of 0-1 mM concentrations and co-administered with pDNA can be exploited as a tool for the control of the efficiency of DNA transfection