I'm about to transform Pichia pastoris with pPICZ alpha and pGAPZalpha with my GOI and because the Invitrogen manual doesn't specify the conditions, I'd like to ask you, what voltage do you use?
Further, while searching the answer online, I found this paper (attached). I just started reading it, but Table 2 caught my attention. They report highest efficiency (transpormants per ug of DNA) with 10 ng of DNA. As the manual say to use 5-10 ug of DNA, that seems awfully low amount. So how much DNA do you use?
Article High efficiency transformation by electroporation of Pichia ...
Dear Tomas,
The paper you refer to (Wu et al) uses a different protocol than the one in the invitrogen manual. This protocol is much more efficient than the one from invitrogen and can be used for library transformations in Pichia.
If you follow the protocol from Wu et al, you can really use 10 ng and have enough transformants to screen for expression. When I used the protocol however, I used 100 ng to have more transformants per electroporation as I did library screening.
If you are interested, I've adapted the method from Wu et al in a step by step protocols article: http://www.ncbi.nlm.nih.gov/pubmed/22886249
I use following conditions on a GenePulser (BioRad): 1.5 kV, capacitance of 25 mFD, extended capacitance of 125 mFD, and resistance of 200 Ohm.
Hope this is of any help,
Best regards,
Kristof
Dear Kristof,
thanks for the answer.
Is it really that different? To me it seems rather the same as the Invitrogen manual for Pichia electroporation. I'll check your paper.
Electroporation conditions for pichia genomic integration:
Voltage : 2000V
Capacitance : 25µF
Resistance : 200R
Cuvette : 2mm (Bio-Rad)
All the best
Dear Tomas,
The difference is in the treatment of the cells with lithium acetate and DTT, for the rest the protocol is rather similar, but it is that treatment that increases the efficiency.
@Kristof
right. I didn't consider that, as that was what they were reporting.
So when the cells are pre-treated with LiAc and DTT, sufficient amount of DNA is around 10 ng, and without treatment one should use 5-10 ug? That's the whole difference?
That is indeed the difference, although one can use 1 µg of DNA with the invitrogen protocol (not needed to have the 5-10µg) but with the pre-treatment the amount of DNA can indeed be lowered to 10 ng
just note that transformation with 10 ng DNA is most efficient (most CFU/ µg DNA), but more DNA can be added to obtain more CFU per transformation
Yes, I'm aware of that. It's just that with less DNA per transformation you can make more transformations and thus increase the number of colonies ;)
It's just I got last time like 10 colonies per transformation so I hope it won't be like that this time.
So I did the transformation on Friday, I tried both with and w/o the LiAc+DTT treatment.
Unfortunately, my restriction of the empty vectors didn't work. I have no idea, since it worked fine with the vectors with my gene.
I transformed around 3-10 ug/transformation (based on nanodrop readings), but I got almost no colonies. And those few colonies that are there, I doubt they are yeasts, because it seems like my selection didn't work well (and I used 150 mg/l zeocin), because I got some small colonies even on the plates with yeast w/o plasmid. But the transformed yeast were viable because when I plated 20 ul on plate w/o zeocin, I got many colonies.
I'll try to check at least those few colonies, whether they are actually transformed Pichia, but I'm sceptical. What could be the problem?
Dear Tomas,
Was the DNA desalted?
Is the Pichia strain you used His+ or did the plates contain His?
It should be the wild-type X-33 and the plates were YPDS (yeast extract, peptone, glucose, sorbitol).
The DNA after digestion was mixed with phenol/chloroform/IAA, then with chloroform and then precipitated first with 96% EtOH and washed with 70% EtOH. But since most of them was OK with 3 ul for the electroporation, I don't think that would be a problem.
Im doing transformations of Pichia, and I use this method:. I let Pichia grow up to 1 DO 600nm in 50 mL, and then do three washes with 25 mL of 1M sorbitol and at the last wash resuspend the pellet in 0.5-0.2 mL of 1M Sorbitol.. The transformation is as follow: Take 80 uL of Pichia and add 1ug of DNA, (by experience it seems that 1ug of linearized plasmid or less works better) put them in the cuvette and let it rest for around 5 minutes. After, u have to do a pulse with 1.5 kV, 25 uF and 186 ohms and resuspend quickly in 1ml of 1M sorbitol.. Then plate the transformant in the appropiate media.
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