Hello everyone,
I am once again transforming yeast Pichia pastoris after many years and have troubles again.
I tried protocol by Kumar (2019)
Article Simplified protocol for faster transformation of (a large nu...
because it seemed nice that it doesn't use large volumes of culture etc. However, there are few details missing, which I tried to fill in with my alleged experience and with other protocols (namely Wu and Letchworth 2004) Article High efficiency transformation by electroporation of Pichia ...
First problem was with my DNA. Although I got nice 500 ng/ul from plasmid purification from bacteria, after digestion and purification, I got only 4-7ng/ul, so I lost like 95% of my DNA somewhere :-/ Thus, I used only about 50 ng of DNA per 40ul of yeast suspension (no carrier DNA used), while the protocol says to use something about 300-500 ng.
However, I encountered a little drawback with the electroporator, which has "advanced" settings, but not for capacitance or resistance, which all protocols mention, but for length of pulse, # of pulses, interval and polarity.
Unlike all other electroporators, this one has maximum length of pulse 200 us! At first, I was going for 25 pulses to get 5 ms, what all protocols mention, but then I realized, that the interval has minimum of 100 ms, which is much longer, so maybe it would not be so good for the yeast? So I went for 5 pulses in the end.
Of course, I got no colonies. So now, I'm wondering, what went wrong. Was it the yeast preparation or the electroporation? I got colonies with water-transformed yeast on YPDS (without Zeocin)
Where would you see the problem? Do you think it's the electroporator? What settings would you recommend? Or shall I play it safe and go for heat shock?
Thank you.
Short update: I tried transformation with 25 pulses to make up 5 ms and in 3 cases out of 3 it caused arc, so that's probably not the way.
I have always followed the Wu and Letchworth 2004 protocol and always had success with it. Never tried the Kumar protocol. Although the Kumar protocol seems to be easier/faster, I would still use the Wu protocol (if it's not broke, don't fix it).
Regarding the electroporator:
I think you need to find a different/simpler electroporator. Specifically one that will you allow you to program/select voltage (kV). The kV needed for Pichia transformation is around 2kV and the time of 5ms. You won't achieve either of these but if you get in the range of 1.8kV and 4.8ms, the transformation efficiency should be fairly high.
I always used a BioRad micropulser electroporator (see attached manual). It has preprogrammed settings for different microbe species (page 9/10) and what the settings are. It's simple, but works.
Dear Prof Masi,
thank you for your answer. I apologize for late reply.
My electroporator enables setting of voltage, but above 500 V, it allows only very short pulses in range of (hundreds of) mikroseconds. The problem is, that it is square wave electroporator, which holds the voltage for the full time, while the classical electroporators' pulse exponentially decay and the time you set or see is for how long you have 1/3 of set voltage (so, for example, if you set 1500 V for 5 ms, it charges to 1500 V, but the voltage decreases immediately and after 5 ms it's at 500 V).
When I understood that, I've set up my electroporator to 500 V for 5 ms and it worked fine.
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