Gongzuo, on Oct 22 2004, 04:56 PM, said:
Hi, you all,
I am a new worker in this area. I am trying to express a protein by using Pichia pastoris. We got the expression kit from Invitrogen. I already have my target gene cloned in pGAPZalphaB and also in pPICZalphaB.
Now my problem is that we cannot get yeast colonies on YPDS-zeocin plates after electroporation of the competent yeasts with the linerized gene construct. I have discussed this problem with seveal professional people, but nobody can find where my problem is. Nobody knows why.
Did anybody get this problem before?
How to make sure that our gene construct can be transfected into the yeast ?
Your answers will be appreciated.
I had some trouble transforming Pichia, and finally managed some good transformants by increasing amount of linealized DNA used in electroporation, Pichia is tricky to transform, you should be using 1-5 ug DNA aprox. Also, minimal amount of resuspending buffer was used (increases concentration). Electroporation is far more efficient than EasyComp kit.
You can check transformants by PCR, using 5AOX and 3AOX primers, purchased from Invitrogen. Good ones would usually give 2 bands: one corresponding to AOX gene and one corresponding to heterologous gene.