I am trying to transform pichia pastoris GS115, but when I run the negative control (the cells without the DNA) it is also growing in the selection medium Minimal Dextrose Medium:1.34% YNB 4 × 10-5 % biotin 2% Dextrose and agar. The vector I am using is pPIC9, which uses the auxotrophy form of selection
When you get the yeast from your freezer stock, does it grown in His- medium as well?
If yes, you have most likely a revertant and you will need new yeast stock.
If not, you probably transform it with HIS4 gene through some contamination.
Also, I assume you tried the transformation repeatedly. If not, it could be just some revertants that grew coincidentally during the preparation of your yeast. However, if it were already in your stock, you should get full plague rather than colonies, because selection should not work at all.
Hi there,
Is your medium actually selective for his4+ clones? The recipe you give is not clear about it...
The recipe of medium selective is composed of: -1.34% YNB
-4× 10^-5 % biotin
-2% dextrose
Samara Alves do you use YNB with or without histidine?
Does your GS115 WT yeast grow on this medium?
Tomás I use YNB without histidine. The GS115 does not grow on this medium
False positive rate is high in Histidine selection. Test more clones with PCR for integration. The other possibility is transferring to an antibiotic plate then screen colonies, positive rate in antibiotic pressure is higher
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