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Electroporation
Basics - Oversimplified Electroporation is a powerful and versatile technique for permealizing cellular membranes to polar molecules, such as DNA, proteins, carbohydrates etc. An electrical field in the order of 0.05-10 kV/cm is briefly (~1-100ms) applied to the cells, causing buildup of the trans-membrane potential of the cell, and leading to the formation of pores in the cellular membranes [1,2]. The mechanism of membrane disruption is not fully understood, but it appears that while the membrane pores form within milliseconds, it takes up to a few minutes for the cells to recover membrane integrity. Consequently, the optimal electroporation conditions are always a tradeoff between maximizing membrane permeability and minimizing cell damage due to leakage. Two types of electrical pulses are currently in use for electroporating cells. In an exponential pulse, the initial electrical field between the electrodes of the cuvette is allowed to decay exponentially, at a rate that is determined by the resistivity of the sample and electrical circuit (Ohms). The overal pulse duration and "strength" also depend on the applied field (Volts) and capacitance (microFarads, uF). These three parameters are controlled to set up an exponential pulse protocol. (In older instruments only the voltage and capacitance can be controlled and the resistivity is simply that of the sample). In a square wave pulse, the electrical field is held almost constant, typically for a few milliseconds, before being dropped to zero. This generally has a milder effect on the cells. The relevant parameters in a square wave pulse are the field (V) and duration (ms). Frequently, electroporation is overlooked as an option for transfection, because it appears technically demanding, and because of the dizzying abundance of alternative transfection methods and reagents offered on the market. And of course, the high cost of electroporation instruments does help either. However, once the protocols have been set up, it offers superior efficiency and versatility to all other methods, especially for primary cell membranes, which are often impervious to chemical transfection.
Omni Universal
Electroporation Solution(TM): Understated Perhaps by serendipitous accident, or perhaps on purpose, Omni Transfection pioneered the use of electroporation additives that help cellular membranes recover after the electric shock. OMNI solution works with a wide range of (we hope, with all) mammalian cell types, and is compatible with all electroporation instruments and protocols. It is especially well-suited for electroporation of siRNA, DNA, and siRNA/DNA mixtures in primary cells and other difficult to transfect cell lines.
Protocol 1. Pre-warm 6-well plates with 3 ml of culture medium per well. Pre-warm OMNI Solution to 37oC. 2. Suspend 5x105 cells in 100 µl OMNI Solution (0.2 cm gap cuvette). Add 0.5-5 µg DNA or 0.1-1 nmol siRNA and tap 10 times to mix. 3. Electroporate following the instrument manufacturer's protocol. A comprehensive list of electroporation conditions tested in various mammalian cell types is available from the BTX website (Registration may be required on the main page before this page can be accessed): http://www.btxonline.com/applications/ protocols/mammalian/Default.asp 4. After pulsing add 500 µl warm medium to the cuvette, then transfer the cells onto the pre-warmed plate. (In our experience so far, a few minutes delay between electroporation and adding the warm media has no effect on cell viability, but this may vary between cell types).
Optimization / Troubleshooting 1. If cell survival rate is low, decrease the field (V), capacitance( µF), or pulse duration, or replace exponential pulses with square wave (sw) pulses.
3. To develop your
own protocol, start with one of the following conditions for 'easy' cell
types such as HeLa or 293: a) Exponential pulse @ 0.65 kV/cm, 500 µF;
b) Exponential pulse @ 1.5 kV/cm, 25 µF; c) 25 ms square wave pulse
@ 0.55 kV/cm. Continue with steps (1) and (2). 4. Do email us with questions - info@omnitransfection.com NOTE: When optimizing electroporation conditions using DNA plasmid expressing a reporter protein (e.g. GFP), differences between promoters controlling the reporter gene may lead to the protein being underexpressed in certain cell types, giving a false negative result. If a surprisingly low transfection efficiency is observed, we recommend trying a different reporter construct.
Reviews on electroporation [1] Gehl, J. Review: Electroporation: theory and methods, perspectives for drug delivery, gene therapy and research. Acta Physiol. Scand. 2003, 177, 437. [2] Escoffre, J. M. et. al. Membrane perturbation by an external electric field: a mechanism to permit molecular uptake. Eur. Biophys. J. 2007, 36, 973.
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