Hi everyone,
Recently I failed several times on transforming Pichia pastoris X-33 strain using electroporation.
The vector I was using was pGAPZ alpha a from Invitrogen.
I also followed the protocol provided from Invitrogen and I have a question about strain cultivation.
I grow P. pastoris in 5 mL of YPD medium broth for overnight at 30 degree Celcius,
then inoculate 500 mL of YPD medium in a 2L flask with 0.5 mL overnight culture,
grow overnight again until OD600= 1.3 - 1.5.
After washing and suspended in 1.5 mL of 1M sorbitol, the suspension was very viscous.
Eventually, I failed the transformation as no Zeocin-resistant colonies grow on the agar plate.
I wonder if the viscosity is the main reason of failure.
Thank you very much.
Follow this protocol and the transformation will work. Use 3ng of DNA (as it shows in the paper) for the electroporation. Also, you need to dilute the cells (1:10, 1:20, 1:50, 1:100) in order to get an accurate OD600 reading otherwise your cells/ml calculation will be wrong and the transformation won't work. It will work for ANY strain of Pichia.
Tom Masi
As per recommendations from invitrogen, 5 to 10 micrograms of linearized DNA should be used. How we can correlate 3 ng with 5 micrograms?
Hi everyone
Can someone guide me that the X33 and SMD1168H strains of yeast are zeocine resistant or not?
I use these strains for transformation of linearized human gene and only cells with no gene used as control but I found the same colonies on the YPDS plates containing zeocine as was found on the cells with genes.
Thanks in advance
Munir Ahmad X-33 strain without any transformed vector should be non-zeocin resistant. Did you use the strain transformed with empty vector instead?
- Badges
- Science topic