I have a question. I have been using the BioRad Gene Pulser in the past and have been able to get it to work for my purposes of adding viral RNA to mammalian cells. However the time constant recently changed greatly and I am confused as to what could cause this and if it negatively affected my experiment. I electroporated BHK21 cells that were suspended in 800ul of PBS in a 4mm cuvette at a concentration of 1x10^7 cells/ml. The settings using the exponential protocol were 850V, 25 capacitance, and infinite resistance. 3 pulses were applied and the usual time constant is between 0.3-0.5. However now the time constant is around 6553 ms. Why is there this dramatic increase? Have I completely fried my cells? What could I do in the future to prevent this? Any help in the matter would be greatly appreciated.
Dear Jasmine
Your time-constant value of 6'553ms means that the voltage you apply with one pulse takes 6.5 seconds to be reduced to one third of the applied 850V.
The time constant is calculated like this: T=RC
Where T is your time-constant, R is the resistance (infinite in your case) and C is the capacitance.
First you might want to determine the health of your cells, otherwise I would go for optimization of the resistance parameter, if you had good experience with your capacitance value.
Frying your cells, in my opinion, is rather caused by arching due the cell sample being too conductive (very low time-constant values). Your case appears to be one of too little conductiveness, indicating a very inefficient pore-formation process by the electric pulse. Or, if this problem came out of the blue and none of the settings have been changed, your electroporator may need maintainance.
Cheers
Marc
Thank you for the response. This value did come out of the blue as we have used these settings previously and everything worked. According to BioRad it was due to the shockpod of the electroporator being broken.
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