I am following some electroporation protocols found in the literature.
Using Genepulser X cell from BioRad and cuvettes 0.2 cm (2mm)
The V (voltage) in the instrument is from 160V to 2500V
How to convert 2.5 kv/cm-1 to the V ? multiply by 1000?
what does cm-1 means? is it 0.1 cm cuvett?
So if I am using 0.2 cm cuvettes and 2.5 kv, is it 2500V / 2 is 1250 V in 2 mm cuvett?
The "Voltage" is what the instrument provides across the electrodes, you can choose that youself. kv/cm-1 means kiloVolts per cm, in other words you have 1000 Volts across 1 cm as "field strenght". So if you use a cuvette with built-in electrodes 2mm apart, and you would like to have 1kv/cm-1 as field strength, you would have to set your machine to 200 Volts. Commercial electroporation cuvettes have built-in electrodes that are typically 1mm, 2mm or 4 mm apart. 1mm is for electroporation of bacteria, 2mm is usually for yeast, and 4 mm is for plant and mammalian cells.
2.5 kv/cm-1 is a high field strength, usually used for bacteria. If you have 1mm cuvettes (0.1 cm), you need to set the voltage to 250Volts in order to obtain 2.5 kv/cm-1 field strength. In my lab we electroporate plant protoplasts, using 4mm cuvettes and 170 Volts (so this is a field strength of 0.425kv/cm-1).
For electroporations, also the capacity is important. We use a complete exponential discharge of 1000 Farad for our plant protoplasts, but for bacterial electroporation at higher field strength the capacity must be much lower than that, otherwise you will boil your cells.
Best wishes,
Jurgen
We have a BioRad Gene Pulser. The settings for E. coli are 200 Ohms (pn a capacitance extender), 25 microFd. Under Volts (set using the up/down arrows), the setting should be 1.8 kV. Hold two red buttons in until you hear a beep, the release. Check Time Constant (on button pad/display). For E. coli the time constant should be between 4.0 and 5.0. Higher than 5.0 means the pores were open too ,long and your cells probably died, or at least a large portion of them. Less than 4.0 means the pores were open too briefly to allow efficient uptake of DNA.
also check the manual, it has settings suggestions for different organisms. Our manual also has explanations in more detail about the physics and math involved with the different parameters.
Pops or crackles are evidence that your DNA is not clean enough. Also be sure to wipe the outside of your ice-chilled cuvette (we use a1 mm gap for E. coli) with a clean, dry tissue before electroporation to remove any condensation.
How many cells can be electroporated in an electroporation cuvette
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