transformation into pichia pastoris - no transformants with pPICZAalpha (May/22/2006 )

Hi there,

my problems concerns transformation of pPICZalpha into P. pastoris GS115. I used 1g purified, linearized DNA. But I do not get transformants. So far I used vector without any heterologous genes to avoid toxic effects. I tested media components, competent cells and electroporation settings with pIC9 and got several hundres positiv clones.

Any ideas about that?
Anyone got the same problem?

Thanks for answers!

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What media did you use. I once had the same problem, I don't remember the names of the plates/medium, but I could not get transformants on the "YPD-like zeocin plates", but I got them on the other (RD?) plates with zeocin. I do remember that the manual tells you to use the "YPD-like" plates. I found it when I did a transformation with PIC9 on both plates, it probably had something to do with the peptone.

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Thanks for your reply. I used YPDS +Zeocin (YDP+Sorbitol plates) as described by the manual. For Pic9 I used YNB (for auxothrophie selection). Is it that what you refered to? I will try this.

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Thanks for your reply. I used YPDS +Zeocin (YDP+Sorbitol plates) as described by the manual. For Pic9 I used YNB (for auxothrophie selection). Is it that what you refered to? I will try this.
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This is what I've used:
RD (Regeneration Dextrose) medium consisted of 1 M sorbitol, 2% dextrose, 1.34 % Yeast Nitrogen Base (10X YNB is 13.4% is 134 gram yeast nitrogen base with ammonium sulfate and without amino acids (Difco) in 1000 ml water), 0.00004% Biotin (0.02% is 20 mg biotin (Fluka) in 100 ml water),1X Amino Acids (100X is 500 mg each of L-glutamic acid, L-methionine, L-lysine, L-leucine and L-isoleucine (Fluka) in 100 ml water).

And then with zeocin added.
I used the SMD1168H strain, so I did not added his.

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This is what I've used:
RD (Regeneration Dextrose) medium consisted of 1 M sorbitol, 2% dextrose, 1.34 % Yeast Nitrogen Base (10X YNB is 13.4% is 134 gram yeast nitrogen base with ammonium sulfate and without amino acids (Difco) in 1000 ml water), 0.00004% Biotin (0.02% is 20 mg biotin (Fluka) in 100 ml water),1X Amino Acids (100X is 500 mg each of L-glutamic acid, L-methionine, L-lysine, L-leucine and L-isoleucine (Fluka) in 100 ml water).

And then with zeocin added.
I used the SMD1168H strain, so I did not added his.
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Thanks, that is really helpfull

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