I will be performing electroporation on Pichia to express a recombinant protein. Is it possible to use 0.1cm cuvette (Frequently used for E.coli or other bacteria) rather than 0.2 cm cuvette? Will there be any adverse effects if I were to use 0.1cm cuvette? Has anyone tried this?
Of course you can. However, you need to optimize the experiment. Give you a reference protocol, based on my experiment, 0.1cm gap, 750V, 25μF, 200ohm, 5ms.
The transformation efficiency is directly related to the method that you prepara the competent cell, generally, 10^4-10^5/μg DNA is favorable.
Of course you can. However, you need to optimize the experiment. Give you a reference protocol, based on my experiment, 0.1cm gap, 750V, 25μF, 200ohm, 5ms.
The transformation efficiency is directly related to the method that you prepara the competent cell, generally, 10^4-10^5/μg DNA is favorable.
Yes, it does affect the electroporation if the cuvette gap width is different, due to the changes in the electrical field (E) during the electroporation. If you are using GenePulser Xcell electroporation system, the technical manual recommends to use the formula, E(electrical field strength, kV/cm) = V (Voltage, kV) /d (cuvette gap width cm). By substituting the E and d value, then you can possible know the input voltage to be used in your experiment. Both 0.1 and 0.2cm cuvettes will respectively give different input voltage with the same electrical field strength. Hope this will help. Goodluck!:)
Hello Min Han,
Thank you very much for your input. I will be trying out electroporation for my Pichia somewhere next week using Zhaowei's given voltage. Will definitely update you on the outcome.
Thanks
If your Pichia competent cells and DNA were prepared correctly and clean enough, you can use higher voltage, up to 1300 V (25 uF/200 ohm), and get higher yield of transformants with 1 mm cuvettes even with low volume of the transformation mixture - 30 - 50 ul. However if an arch is observed, you better reduce the voltage (or clean either the cells or DNA from traces of salt).
Hi Guys,
I have successfully electroporated the linarized plasmid using 0.1cm cuvette and have confirmed the presence of my targeted gene via PCR.
Thank you.
Hi Wei Li..below is the detailed protocol using 0.1cm cuvette:
I tried 3 different voltages (750V; 1.0kV and 1.2kV). And I obtained positive transformations with all the voltage applied. Briefly, I cultured 10mL of my cells as my initial culture fr 24-48 hours. Using the initial culture, I then inoculated into 100mL of my media and incubate further until my OD600 reached 1.5. Once the OD reached 1.5, I centrifuged the cells and washed them 3-4X with 1M sorbitol solution (Make sure that all your centrifugation step is performed at cold temperature as the temperature plays a critical role in softening the cell wall of the cells-read it somewhere, but not sure if this is true?) Also make sure that you desalt the plasmid DNA/cells properly, as the residual carry over of salt can cause arch when electroporating.
After the washing step, I then re-suspended the cells in 300uL of 1M-chilled Sorbitol and kept the cells on ice. For transformation, I used 100uL of the freshly prepared competent cells and mixed well with ~10ug (Some ppl have also tried with jus 1ug of plasmid DNA in my lab) of my linearized plasmid. I further incubated the cells on ice for about 5 minutes together with the cuvette and after that performed electroporation with 25uF, 186-200ohm.
Best of luck
Saran
hi saran
can you please tell me what was your transformation efficiency? did you transform a library?
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